Endophytes, associated compositions, and methods of use thereof

ABSTRACT

Materials and methods for improving plant traits and for providing plant benefits are provided. In some embodiments, the materials, and methods employing the same, can comprise endophytes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/752,829, filed Jun. 26, 2015, (pending) which is acontinuation-in-part of International Application No. PCT/US2014/044427,filed Jun. 26, 2014 (pending) which is hereby incorporated in itsentirety by reference. U.S. patent application Ser. No. 14/752,829,filed Jun. 26, 2015, (pending) claims the benefit of U.S. ProvisionalApplication No. 62/017,796, filed Jun. 26, 2014, and U.S. ProvisionalApplication No. 62/017,809, filed Jun. 26, 2014, and U.S. ProvisionalApplication No. 62/017,816, filed Jun. 26, 2014, and U.S. ProvisionalApplication No. 62/017,813, filed Jun. 26, 2014, and U.S. ProvisionalApplication No. 62/017,815, filed Jun. 26, 2014, and U.S. ProvisionalApplication No. 62/017,818, filed Jun. 26, 2014, each of which is herebyincorporated in its entirety by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Sep. 2, 2015, is named30696US_CRF_sequencelisting.txt, and is 2,970,000 bytes in size.

FIELD OF THE INVENTION

This invention relates to compositions and methods for improving thecultivation of plants, particularly agricultural plants. For example,this invention describes beneficial bacteria and fungi that are capableof living in a plant, which may be used to impart improved agronomictraits to plants. The disclosed invention also describes methods ofimproving plant characteristics by introducing such beneficial bacteriaand/or fungi to those plants. Further, this invention also providesmethods of treating seeds and other plant elements with beneficialbacteria and/or fungi that are capable of living within a plant, toimpart improved agronomic characteristics to plants, particularlyagricultural plants.

BACKGROUND

Agriculture faces numerous challenges that are making it increasinglydifficult to provide food, materials, and fuels to the world'spopulation. Population growth and changes in diet associated with risingincomes are increasing global food demand, while many key resources foragriculture are becoming increasingly scarce. By 2050, the FAO projectsthat total food production must increase by 70% to meet the needs of thegrowing population, a challenge that is exacerbated by numerous factors,including diminishing freshwater resources, increasing competition forarable land, rising energy prices, increasing input costs, and thelikely need for crops to adapt to the pressures of a more extreme globalclimate. The need to grow nearly twice as much food in more uncertainclimates is driving a critical need for new innovations.

Today, crop performance is optimized via of technologies directedtowards the interplay between crop genotype (e.g., plant breeding,genetically-modified (GM) crops) and its surrounding environment (e.g.,fertilizer, synthetic herbicides, pesticides). While these paradigmshave assisted in doubling global food production in the past fiftyyears, yield growth rates have stalled in many major crops and shifts inthe climate have been linked to production declines in important cropssuch as wheat. In addition to their long development and regulatorytimelines, public fears of GM-crops and synthetic chemicals haschallenged their use in many key crops and countries, resulting in acomplete lack of acceptance for GM traits in wheat and the exclusion ofGM crops and many synthetic chemistries from European markets. Thus,there is a significant need for innovative, effective, andpublically-acceptable approaches to improving the intrinsic yield andresilience of crops to severe stresses.

Like humans, which benefit from a complement of beneficial microbialsymbionts, plants have been purported to benefit somewhat from the vastarray of bacteria and fungi that live both within and around theirtissues to support their health and growth. Endophytes are fungal orbacterial organisms that live within plants. Bacterial and fungalendophytes appear to inhabit various host plant tissues and have beenisolated from plant leaves, stems, or roots.

A small number of these symbiotic endophyte-host relationships have beenpurported in limited studies to provide agronomic benefits to model hostplants within controlled laboratory settings, such as enhancement ofbiomass production (i.e., yield) and nutrition, increased tolerance tostress such as drought, and/or pests. Yet, such endophytes have beendemonstrated to be ineffective in conferring benefits to a variety ofagriculturally-important plants; as such, they do not adequately addressthe need to provide improved yield and tolerance to environmentalstresses present in many agricultural situations for such crops.

Thus, there is a need for compositions and methods of providingagricultural crops with improved yield and resistance to variousenvironmental stresses. Provided herein are novel compositions ofbacterial and fungal endophytes and synthetic endophyte-plantcompositions based on the analysis of the key properties that enhancethe utility and commercialization of an endophytic composition.

SUMMARY OF THE INVENTION

The disclosures of PCT/US2014/044427, filed Jun. 26, 2014, and U.S.application Ser. No. 14/316,469, filed Jun. 26, 2014, are incorporatedby reference in their entirety, including the sequence listingcontaining SEQ ID NOs: 1-1448.

The present invention is based on the surprising discovery that a numberof bacterial and fungal taxa of endophytes microbes are conserved acrossdiverse species and/or cultivars of agricultural plants, and can bederived therefrom and heterologously associated with diverse newcultivars to provide benefits. The present invention is also based onthe discovery that a plant element of a plant can be effectivelyaugmented by coating its surface with such endophytes in an amount thatis not normally found on the plant element. The endophytes can beisolated from inside the same plant or a different plant, or from insidea part or tissue of the same plant or different plant. The plant elementthus coated with the endophyte can be used to confer improved agronomictrait or traits to the seed or the plant that is grown from the plantelement.

The inventors have postulated that attempts to select for cultivars withcertain improved traits and alterations in the environmental andchemical conditions of agriculture have led to the inadvertent loss ofmicrobes in modern varieties that can provide beneficial traits toagricultural plants. The present invention is based on the surprisingdiscovery that many modern cultivars of agricultural plants displaystriking distinctions in their microbial communities when compared withancestral varieties. The present invention is also based on theobservation that, in some cases, providing the microbial taxa present insuch ancestral cultivars but are absent or underrepresented in modernvarieties can lead to dramatic improvements in a number of agronomictraits in the modern cultivars.

SUMMARY

Described herein are methods for preparing an agricultural seedcomposition comprising contacting the surface of a plurality of seedswith a formulation comprising a purified microbial population thatcomprises at least two endophytes that are heterologous to the seed. Thefirst endophyte is capable of metabolizing at least one of D-alanine,D-aspartic acid, D-serine, D-threonine, glycyl-L-aspartic acid,glycyl-L-glutamic acid, glycyl-L-proline, glyoxylic acid, inosine,L-alanine, L-alanyl-glycine, L-arabinose, L-asparagine, L-aspartic acid,L-glutamic acid, L-glutamine, L-proline, L-serine, L-threonine,tyramine, uridine, proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, and salicin and theendophytes are present in the formulation in an amount capable ofmodulating a trait of agronomic importance, as compared to isolineplants grown from seeds not contacted with the formulation.

Also described herein are method for preparing an agricultural seedcomposition, comprising contacting the surface of a plurality of seedswith a formulation comprising a purified microbial population thatcomprises at least two endophytes that are heterologous to the seed. Thefirst endophyte is capable of at least one function or activity selectedfrom the group consisting of auxin production, nitrogen fixation,production of an antimicrobial compound, mineral phosphatesolubilization, siderophore production, cellulase production, chitinaseproduction, xylanase production, and acetoin production and theendophytes are present in the formulation in an amount capable ofmodulating a trait of agronomic importance, as compared to isolineplants grown from seeds not contacted with the formulation.

Also described are methods of improving a phenotype during water limitedconditions of a plurality of host plants grown from a plurality ofseeds, comprising treating the seeds with a formulation comprising atleast two endophytes that are heterologous to the seeds. The firstendophyte is capable of metabolizing at least one of D-alanine,D-aspartic acid, D-serine, D-threonine, glycyl-L-aspartic acid,glycyl-L-glutamic acid, glycyl-L-proline, glyoxylic acid, inosine,L-alanine, L-alanyl-glycine, L-arabinose, L-asparagine, L-aspartic acid,L-glutamic acid, L-glutamine, L-proline, L-serine, L-threonine,tyramine, uridine, proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, and salicin. Thephenotype improvement is selected from the group consisting of: diseaseresistance, heat tolerance, cold tolerance, salinity tolerance, metaltolerance, herbicide tolerance, chemical tolerance, improved nitrogenutilization, improved resistance to nitrogen stress, improved nitrogenfixation, pest resistance, herbivore resistance, pathogen resistance,increased yield, health enhancement, vigor improvement, growthimprovement, photosynthetic capability improvement, nutritionenhancement, altered protein content, altered oil content, increasedbiomass, increased shoot length, increased root length, improved rootarchitecture, increased seed weight, altered seed carbohydratecomposition, altered seed oil composition, number of pods, delayedsenescence, stay-green, and altered seed protein composition, increaseddry weight of mature seeds, increased fresh weight of mature seeds,increased number of mature seeds per plant, increased chlorophyllcontent, increased number of pods per plant, increased length of podsper plant, reduced number of wilted leaves per plant, reduced number ofseverely wilted leaves per plant, and increased number of non-wiltedleaves per plant.

The seed or plant can be a dicot, e.g., soybean, cotton, tomato andpepper or a monocot, e.g., corn, wheat, barley and rice. In someembodiments, the seed is a transgenic seed.

The methods described herein include a first endophyte and a secondendophyte. The first endophyte and/or the second endophyte can be, e.g.,a bacterial endophyte or, e.g., a fungal endophyte. Examples ofbacterial endophytes include, e.g., those from a genus selected from thegroup consisting of: Acidovorax, Agrobacterium, Bacillus, Burkholderia,Chryseobacterium, Curtobacterium, Enterobacter, Escherichia,Methylobacterium, Paenibacillus, Pantoea, Pseudomonas, Ralstonia,Saccharibacillus, Sphingomonas, and Stenotrophomonas. In someembodiments, the bacterial endophyte has a 16S rRNA sequence that is atleast 95% identical to a sequence selected from the group consisting of:SEQ ID NOs: 3588, 3589, 3590, 3591, 3592, 3593, 3594, 3595, 3596, 3598,3599, 3600, 3601, 3603, 3604, 3606, 3607, 3608, 3609, 3619, 3620, 3621,3622, 3623, 3624, 3625, 3626, 3627, 3628, 3629, 3630, 3631, 3632, 3633,3634, 3635, 3636, 3637, 3638, 3639, 3641, 3645, 3646, 3648, 3649, 3651,3652, 3653, 3656, 3663, 3664, 3665, 3666, 3667, 3668, 3669, 3670, 3671.

Examples of fungal endophytes include, e.g., those from a genus selectedfrom the group consisting of: Acremonium, Alternaria, Cladosporium,Cochliobolus, Embellisia, Epicoccum, Fusarium, Nigrospora, Phoma, andPodospora. In some embodiments, the fungal endophyte has an ITS rRNA atleast 95% identical to a sequence selected from the group consisting of:SEQ ID NOs: 3597, 3602, 3605, 3610, 3611, 3612, 3613, 3614, 3615, 3616,3617, 3618, 3640, 3642, 3643, 3644, 3647, 3650, 3654, 3655, 3657, 3658,3659, 3660, 3661, 3662, 3672, 3673, 3674, 3675, 3676, 3677, 3678, 3679,3680, 3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688, 3689, 3690, 3691,3692, 3693, 3694, 3695, 3696, 3697, 3698, 3699, 3700.

In some embodiments, the formulation comprises at least two endophyticmicrobial entities provided in any of Tables 2B, 3B, 4B, and 15.

In some embodiments of the methods described herein, the first endophyteis capable of metabolizing at least two of D-alanine, D-aspartic acid,D-serine, D-threonine, glycyl-L-aspartic acid, glycyl-L-glutamic acid,glycyl-L-proline, glyoxylic acid, inosine, L-alanine, L-alanyl-glycine,L-arabinose, L-asparagine, L-aspartic acid, L-glutamic acid,L-glutamine, L-proline, L-serine, L-threonine, tyramine, uridine,proline, arabinose, xylose, mannose, sucrose, maltose, D-glucosamine,trehalose, oxalic acid, and salicin. In some embodiments of the methodsdescribed herein, the second endophyte is capable of metabolizing atleast one of D-alanine, D-aspartic acid, D-serine, D-threonine,glycyl-L-aspartic acid, glycyl-L-glutamic acid, glycyl-L-proline,glyoxylic acid, inosine, L-alanine, L-alanyl-glycine, L-arabinose,L-asparagine, L-aspartic acid, L-glutamic acid, L-glutamine, L-proline,L-serine, L-threonine, tyramine, uridine, proline, arabinose, xylose,mannose, sucrose, maltose, D-glucosamine, trehalose, oxalic acid, andsalicin.

The methods described herein include a formulation. In some embodiments,the formulation comprises the purified microbial population at aconcentration of at least about 10̂2 CFU/ml or spores/ml in a liquidformulation or about 10̂2 CFU/gm or spores/ml in a non-liquidformulation. In some embodiments, the formulation further comprises oneor more of the following: a stabilizer, or a preservative, or a carrier,or a surfactant, or an anticomplex agent, or any combination thereofand/or one or more of the following: fungicide, nematicide, bactericide,insecticide, and herbicide.

In some embodiments, the methods described herein modulate a traitagronomic importance. The trait of agronomic importance can be, e.g.,disease resistance, drought tolerance, heat tolerance, cold tolerance,salinity tolerance, metal tolerance, herbicide tolerance, chemicaltolerance, improved water use efficiency, improved nitrogen utilization,improved resistance to nitrogen stress, improved nitrogen fixation, pestresistance, herbivore resistance, pathogen resistance, increased yield,increased yield under water-limited conditions, health enhancement,vigor improvement, growth improvement, photosynthetic capabilityimprovement, nutrition enhancement, altered protein content, altered oilcontent, increased biomass, increased shoot length, increased rootlength, improved root architecture, increased seed weight, altered seedcarbohydrate composition, altered seed oil composition, number of pods,delayed senescence, stay-green, and altered seed protein composition.

The methods described herein can include at least one endophyte capableof localizing in a plant element of a plant grown from said seed, saidplant element selected from the group consisting of: whole plant,seedling, meristematic tissue, ground tissue, vascular tissue, dermaltissue, seed, leaf, root, shoot, stem, flower, fruit, stolon, bulb,tuber, corm, keikis, and bud.

In some embodiments, the methods described herein further includeplacing the plurality of seeds into a substrate that promotes plantgrowth, including but not limited to soil. For examples, the seeds areplaced in the soil in rows, with substantially equal spacing betweeneach seed within each row.

Also described herein is a plant derived from the agricultural seedpreparation of the methods described herein, wherein said plantcomprises in at least one of its plant elements said endophytes, and/orwherein said progeny comprises in at least one of its plant elementssaid endophytes. Also described herein is a plurality of seedcompositions prepared according to the methods described herein, whereinsaid seed compositions are confined within an object selected from thegroup consisting of: bottle, jar, ampule, package, vessel, bag, box,bin, envelope, carton, container, silo, shipping container, truck bed,and case.

Described herein are methods for preparing a seed comprising anendophyte population, said method comprising applying to an exteriorsurface of a seed a formulation comprising an endophyte populationconsisting essentially of an endophyte comprising a 16S rRNA or ITS rRNAnucleic acid sequence at least 95% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1-3700; methods fortreating seedlings, the method comprising contacting foliage or therhizosphere of a plurality of agricultural plant seedlings with a seed aformulation comprising an endophyte population consisting essentially ofan endophyte comprising a 16S rRNA or ITS rRNA nucleic acid sequence atleast 95% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 1-3700; and growing the contacted seedlings;methods for modulating a plant trait comprising applying to vegetationor an area adjacent the vegetation, a seed a formulation comprising anendophyte population consisting essentially of an endophyte comprising a16S rRNA or ITS rRNA nucleic acid sequence at least 95% identical to anucleic acid sequence selected from the group consisting of SEQ ID NOs:1-3700, wherein the formulation is capable of providing a benefit to thevegetation, or to a crop produced from the vegetation; and methods formodulating a plant trait comprising applying a formulation to soil, theseed a formulation comprising an endophyte population consistingessentially of an endophyte comprising a 16S rRNA or ITS rRNA nucleicacid sequence at least 95% identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-3700, wherein the formulationis capable of providing a benefit to seeds planted within the soil, orto a crop produced from plants grown in the soil. In some embodiments,the method includes applying or contacting by spraying, immersing,coating, encapsulating, or dusting the seeds or seedlings with theformulation.

Described herein are methods for improving an agricultural trait in anagricultural plant, the method comprising providing a modernagricultural plant, contacting said plant with a formulation comprisingan endophyte derived from an ancestral plant in an amount effective tocolonize the plant and allowing the plant to grow under conditions thatallow the endophyte to colonize the plant, and methods for improving anagricultural trait in an agricultural plant, the method comprisingproviding an agricultural plant, contacting said plant with aformulation comprising an endophyte that is common to at least two donorplant types that is present in the formulation in an amount effective tocolonize the plant, and growing the plants under conditions that allowthe endophyte to improve a trait in the plant. In some embodiments, theendophyte comprises a 16S rRNA or ITS rRNA nucleic acid sequence atleast 95% identical to a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 1-3700. In some embodiments, the methodincludes applying or contacting by spraying, immersing, coating,encapsulating, or dusting the seeds or seedlings with the formulation.

The seed or plant can be a dicot, e.g., soybean, cotton, tomato andpepper or a monocot, e.g., corn, wheat, barley and rice. In someembodiments, the seed is a transgenic seed.

In some embodiments, the endophyte is capable of exhibiting productionof an auxin, nitrogen fixation, production of an antimicrobial,production of a siderophore, mineral phosphate solubilization,production of a cellulase, production of a chitinase, production of axylanase, or production of acetoin, e.g., the endophyte exhibits atleast two of: production of an auxin, nitrogen fixation, production ofan antimicrobial, production of a siderophore, mineral phosphatesolubilization, production of a cellulase, production of a chitinase,production of a xylanase, and production of acetoin. In otherembodiments, the endophyte is capable of metabolizing at least one ofD-alanine, D-aspartic acid, D-serine, D-threonine, glycyl-L-asparticacid, glycyl-L-glutamic acid, glycyl-L-proline, glyoxylic acid, inosine,L-alanine, L-alanyl-glycine, L-arabinose, L-asparagine, L-aspartic acid,L-glutamic acid, L-glutamine, L-proline, L-serine, L-threonine,tyramine, uridine, proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, and salicin. In furtherembodiments, the endophyte is capable of capable of metabolizing atleast two of D-alanine, D-aspartic acid, D-serine, D-threonine,glycyl-L-aspartic acid, glycyl-L-glutamic acid, glycyl-L-proline,glyoxylic acid, inosine, L-alanine, L-alanyl-glycine, L-arabinose,L-asparagine, L-aspartic acid, L-glutamic acid, L-glutamine, L-proline,L-serine, L-threonine, tyramine, uridine, proline, arabinose, xylose,mannose, sucrose, maltose, D-glucosamine, trehalose, oxalic acid, andsalicin.

In some embodiments, the endophyte comprises a nucleic acid sequencethat is at least 97% identical to any nucleic acid provided in Tables1A, 2A, 3A, 4A, 5-14, 16-23, wherein the endophyte is present in theformulation in an amount effective to colonize the mature agriculturalplant. In other embodiments, at least one of the endophytes comprises anucleic acid sequence that is at least 97% identical to any nucleic acidprovided in Tables 1A, 2A, 3A, 4A, 5-14, 16-23, wherein the endophyte ispresent in the formulation in an amount effective to colonize the matureagricultural plant.

The endophyte can be present at a concentration of, for example, atleast 10² CFU or spores/seed on the surface of the seeds aftercontacting.

In some embodiments, the methods described herein modulate a traitagronomic importance. The benefit or agricultural trait can be selectedfrom the group consisting of: increased root biomass, increased rootlength, increased height, increased shoot length, increased leaf number,increased water use efficiency, increased tolerance to low nitrogenstress, increased nitrogen use efficiency, increased overall biomass,increase grain yield, increased photosynthesis rate, increased toleranceto drought, increased heat tolerance, increased salt tolerance,increased resistance to nematode stress, increased resistance to afungal pathogen, increased resistance to a bacterial pathogen, increasedresistance to a viral pathogen, a detectable modulation in the level ofa metabolite, and a detectable modulation in the proteome, relative toreference seeds or agricultural plants derived from reference seeds. Insome embodiments, the benefit or agricultural trait comprises at leasttwo benefits or agricultural traits selected from the group consistingof: increased root biomass, increased root length, increased height,increased shoot length, increased leaf number, increased water useefficiency, increased tolerance to low nitrogen stress, increasednitrogen use efficiency, increased overall biomass, increase grainyield, increased photosynthesis rate, increased tolerance to drought,increased heat tolerance, increased salt tolerance, increased resistanceto nematode stress, increased resistance to a fungal pathogen, increasedresistance to a bacterial pathogen, increased resistance to a viralpathogen, a detectable modulation in the level of a metabolite, and adetectable modulation in the proteome, relative to reference seeds orplants derived from reference seeds. Examples include but are notlimited to increased tolerance to low nitrogen stress or increasednitrogen use efficiency, and the endophyte is non-diazotrophic orincreased tolerance to low nitrogen stress or increased nitrogen useefficiency, and the endophyte is diazotrophic.

In some embodiments, the formulation comprises at least one memberselected from the group consisting of an agriculturally compatiblecarrier, a tackifier, a microbial stabilizer, a fungicide, anantibacterial agent, an herbicide, a nematicide, an insecticide, a plantgrowth regulator, a rodenticide, and a nutrient.

The methods described herein can include contacting the seed or plantwith at least 100 CFU or spores, at least 300 CFU or spores, at least1,000 CFU or spores, at least 3,000 CFU or spores, at least 10,000 CFUor spores, at least 30,000 CFU or spores, at least 100,000 CFU orspores, at least 300,000 CFU or spores, at least 1,000,000 CFU or sporesor more, of the endophyte.

In some embodiments of the methods described herein, the endophyte ispresent in the formulation in an amount effective to be detectablewithin a target tissue of the agricultural plant selected from a fruit,seed, leaf, root or portion thereof. For example, the population isdetected in an amount of at least 100 CFU or spores, at least 300 CFU orspores, at least 1,000 CFU or spores, at least 3,000 CFU or spores, atleast 10,000 CFU or spores, at least 30,000 CFU or spores, at least100,000 CFU or spores, at least 300,000 CFU or spores, at least1,000,000 CFU or spores, or more, in the target tissue. Alternatively orin addition, the endophyte is present in the formulation in an amounteffective to increase the biomass and/or yield of the fruit or seedproduced by the plant by at least 1%, at least 2%, at least 3%, at least5%, at least 10%, at least 15%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 100%, or more, when compared with the fruit or seed of areference agricultural plant. Alternatively or in addition, theendophyte is present in the formulation in an amount effective todetectably increase the biomass of the plant, or a part or a tissue typethereof, e.g., detectably increased by at least 1%, at least 2%, atleast 3%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, or more, when compared with areference agricultural plant. Alternatively or in addition, theendophyte is present in the formulation in an amount effective todetectably increase the rate of germination of the seed, e.g., increasedby at least 0.5%, at least 1%, at least 2%, at least 3%, at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100% ormore, when compared with a reference agricultural plant.

Also described herein are synthetic compositions comprising a purifiedmicrobial population in association with a plurality of seeds orseedlings of an agricultural plant, wherein the purified microbialpopulation comprises a first endophyte capable of at least one of:production of an auxin, nitrogen fixation, production of anantimicrobial, production of a siderophore, mineral phosphatesolubilization, production of a cellulase, production of a chitinase,production of a xylanase, and production of acetoin, or a combination oftwo or more thereof, wherein the first endophyte comprises a 16S rRNA orITS rRNA nucleic acid sequence at least 95% identical to a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 1-3700, andwherein the endophyte is present in the synthetic combination in anamount effective to provide a benefit to the seeds or seedlings or theplants derived from the seeds or seedlings. In some embodiments, theformulation comprises at least two endophytes provided in any of Tables2B, 3B, 4B, and 15.

Also described herein are synthetic compositions comprising a purifiedpopulation in association with a plurality of seeds or seedlings of anagricultural plant, wherein the purified microbial population comprisesa first endophyte wherein the first endophyte is capable of metabolizingat least one of D-alanine, D-aspartic acid, D-serine, D-threonine,glycyl-L-aspartic acid, glycyl-L-glutamic acid, glycyl-L-proline,glyoxylic acid, inosine, L-alanine, L-alanyl-glycine, L-arabinose,L-asparagine, L-aspartic acid, L-glutamic acid, L-glutamine, L-proline,L-serine, L-threonine, tyramine, uridine, proline, arabinose, xylose,mannose, sucrose, maltose, D-glucosamine, trehalose, oxalic acid, andsalicin, wherein the first endophyte comprises a 16S rRNA or ITS rRNAnucleic acid sequence at least 95% identical to a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1-3700, and whereinthe endophyte is present in the synthetic combination in an amounteffective to provide a benefit to the seeds or seedlings or the plantsderived from the seeds or seedlings. In some embodiments, the microbialpopulation further comprises a second endophyte, wherein the first andsecond endophytes are independently capable of metabolizing at least oneof D-alanine, D-aspartic acid, D-serine, D-threonine, glycyl-L-asparticacid, glycyl-L-glutamic acid, glycyl-L-proline, glyoxylic acid, inosine,L-alanine, L-alanyl-glycine, L-arabinose, L-asparagine, L-aspartic acid,L-glutamic acid, L-glutamine, L-proline, L-serine, L-threonine,tyramine, uridine, proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, and salicin, or acombination of two or more thereof. In some embodiments, the twoendophytes are provided in any of Tables 2B, 3B, 4B, and 15

Also described herein are synthetic compositions comprising at least twoendophytes associated with a seed, wherein at least the first endophyteis heterologous to the seed and is capable of production of an auxin,nitrogen fixation, production of an antimicrobial, production of asiderophore, mineral phosphate solubilization, production of acellulase, production of a chitinase, production of a xylanase, andproduction of acetoin, or a combination of two or more thereof, whereinthe endophytes are present in the formulation in an amount effective toprovide a benefit to the seeds or seedlings or the plants derived fromthe seeds or seedlings. In some embodiments, both of the endophytes areheterologous to the seed. In some embodiments, the first and secondendophytes are independently capable of at least one of production of anauxin, nitrogen fixation, production of an antimicrobial, production ofa siderophore, mineral phosphate solubilization, production of acellulase, production of a chitinase, production of a xylanase, orproduction of acetoin, or a combination of two or more thereof. In someembodiments, first endophyte comprises a 16S rRNA or ITS rRNA nucleicacid sequence at least 95% identical to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-3700. In some embodiments,the formulation comprises at least two endophytes provided in any ofTables 2B, 3B, 4B, and 15.

Also described herein are synthetic compositions comprising at least twoendophytes associated with a seed, wherein at least the first endophyteis heterologous to the seed and is capable of metabolizing at least oneof D-alanine, D-aspartic acid, D-serine, D-threonine, glycyl-L-asparticacid, glycyl-L-glutamic acid, glycyl-L-proline, glyoxylic acid, inosine,L-alanine, L-alanyl-glycine, L-arabinose, L-asparagine, L-aspartic acid,L-glutamic acid, L-glutamine, L-proline, L-serine, L-threonine,tyramine, uridine, proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, and salicin, wherein theendophytes are present in the formulation in an amount effective toprovide a benefit to the seeds or seedlings or the plants derived fromthe seeds or seedlings. In some embodiments, both of the endophytes areheterologous to the seed. In some embodiments, the first and secondendophytes are independently capable of metabolizing at least one ofD-alanine, D-aspartic acid, D-serine, D-threonine, glycyl-L-asparticacid, glycyl-L-glutamic acid, glycyl-L-proline, glyoxylic acid, inosine,L-alanine, L-alanyl-glycine, L-arabinose, L-asparagine, L-aspartic acid,L-glutamic acid, L-glutamine, L-proline, L-serine, L-threonine,tyramine, uridine, proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, and salicin, or acombination of two or more thereof. In some embodiments, first endophytecomprises a 16S rRNA or ITS rRNA nucleic acid sequence at least 95%identical to a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 1-3700.

In some embodiments, the synthetic combinations described herein aredisposed within a packaging material selected from a bag, box, bin,envelope, carton, or container. In some embodiments, the syntheticcombinations described herein comprise 1000 seed weight amount of seeds,wherein the packaging material optionally comprises a dessicant, andwherein the synthetic combination optionally comprises an anti-fungalagent.

In some embodiments, the synthetic combinations described hereincomprise a first endophyte that is localized on the surface of the seedsor seedlings; and/or obtained from a plant species other than the seedsor seedlings of the synthetic combination; and/or obtained from a plantcultivar different from the cultivar of the seeds or seedlings of thesynthetic combination; and/or obtained from a plant cultivar that is thesame as the cultivar of the seeds or seedlings of the syntheticcombination.

In some embodiments, the synthetic compositions comprising a purifiedpopulation in association with a plurality of seeds or seedlings of anagricultural plant the microbial population further comprise a secondendophyte, for example, a second microbial endophyte having an 16S rRNAor ITS rRNA nucleic acid sequence less than 95% identical to that of thefirst microbial endophyte. In some embodiments, the first and secondendophytes are independently capable of at least one of production of anauxin, nitrogen fixation, production of an antimicrobial, production ofa siderophore, mineral phosphate solubilization, production of acellulase, production of a chitinase, production of a xylanase, orproduction of acetoin, or a combination of two or more thereof.

In some embodiments, the synthetic combinations described hereincomprise, for example, a first endophyte that is a bacterial endophyte;a first endophyte that is a bacterial endophyte and a second endophytethat is a bacterial endophyte; a first endophyte that is a bacterialendophyte and a second endophyte that is a fungal endophyte; a firstendophyte that is a fungal endophyte; and/or a first endophyte that is afungal endophyte and a second endophyte that is a fungal endophyte.

In the embodiments with a second endophyte, the bacterial endophyte canbe, e.g., of a genus selected from the group consisting of: Acidovorax,Agrobacterium, Bacillus, Burkholderia, Chryseobacterium, Curtobacterium,Enterobacter, Escherichia, Methylobacterium, Paenibacillus, Pantoea,Pseudomonas, Ralstonia, Saccharibacillus, Sphingomonas, andStenotrophomonas; and/or the bacterial endophyte can be one with a 16SrRNA sequence that is at least 95% identical to a sequence selected fromthe group consisting of: SEQ ID NOs: 3588, 3589, 3590, 3591, 3592, 3593,3594, 3595, 3596, 3598, 3599, 3600, 3601, 3603, 3604, 3606, 3607, 3608,3609, 3619, 3620, 3621, 3622, 3623, 3624, 3625, 3626, 3627, 3628, 3629,3630, 3631, 3632, 3633, 3634, 3635, 3636, 3637, 3638, 3639, 3641, 3645,3646, 3648, 3649, 3651, 3652, 3653, 3656, 3663, 3664, 3665, 3666, 3667,3668, 3669, 3670, 3671.

The fungal endophyte can be, e.g., of a genus selected from the groupconsisting of: Acremonium, Alternaria, Cladosporium, Cochliobolus,Embellisia, Epicoccum, Fusarium, Nigrospora, Phoma, and Podospora and/orhave an ITS rRNA at least 95% identical to a sequence selected from thegroup consisting of: SEQ ID NOs: 3597, 3602, 3605, 3610, 3611, 3612,3613, 3614, 3615, 3616, 3617, 3618, 3640, 3642, 3643, 3644, 3647, 3650,3654, 3655, 3657, 3658, 3659, 3660, 3661, 3662, 3672, 3673, 3674, 3675,3676, 3677, 3678, 3679, 3680, 3681, 3682, 3683, 3684, 3685, 3686, 3687,3688, 3689, 3690, 3691, 3692, 3693, 3694, 3695, 3696, 3697, 3698, 3699,3700.

The synthetic combinations described herein can include, for example, afirst endophyte capable of at least two of: production of an auxin,nitrogen fixation, production of an antimicrobial, production of asiderophore, mineral phosphate solubilization, production of acellulase, production of a chitinase, production of a xylanase,utilization of arabinose as a carbon source, and production of acetoin;and/or capable of metabolizing at least two of D-alanine, D-asparticacid, D-serine, D-threonine, glycyl-L-aspartic acid, glycyl-L-glutamicacid, glycyl-L-proline, glyoxylic acid, inosine, L-alanine,L-alanyl-glycine, L-arabinose, L-asparagine, L-aspartic acid, L-glutamicacid, L-glutamine, L-proline, L-serine, L-threonine, tyramine, uridine,proline, arabinose, xylose, mannose, sucrose, maltose, D-glucosamine,trehalose, oxalic acid, and salicin, or a combination of two or morethereof.

The synthetic combinations described herein can include, for example, afirst endophyte comprises a nucleic acid sequence that is at least 97%identical to any nucleic acid provided in Tables 1A, 2A, 3A, 4A, 5-14,16-23.

The synthetic combinations described herein can include, for example,first endophytes present in an amount of at least about 100 CFU orspores, at least 300 CFU or spores, at least 1,000 CFU or spores, atleast 3,000 CFU or spores, at least 10,000 CFU or spores, at least30,000 CFU or spores, at least 100,000 CFU or spores, at least 300,000CFU or spores, at least 1,000,000 CFU spores per seed.

In some embodiments, the synthetic combinations described hereincomprise a benefit selected from the group consisting of increased rootbiomass, increased root length, increased height, increased shootlength, increased leaf number, increased water use efficiency, increasedoverall biomass, increase grain yield, increased photosynthesis rate,increased tolerance to drought, increased heat tolerance, increased salttolerance, increased resistance to nematode stress, increased resistanceto a fungal pathogen, increased resistance to a bacterial pathogen,increased resistance to a viral pathogen, a detectable modulation in thelevel of a metabolite, and a detectable modulation in the proteomerelative to a reference plant. In some embodiments, the syntheticcombinations described herein comprise at least two benefits selectedfrom the group consisting of increased root biomass, increased rootlength, increased height, increased shoot length, increased leaf number,increased water use efficiency, increased tolerance to low nitrogenstress, increased nitrogen use efficiency, increased overall biomass,increase grain yield, increased photosynthesis rate, increased toleranceto drought, increased heat tolerance, increased salt tolerance,increased resistance to nematode stress, increased resistance to afungal pathogen, increased resistance to a bacterial pathogen, increasedresistance to a viral pathogen, a detectable modulation in the level ofa metabolite, and a detectable modulation in the proteome, relative to areference plant.

In some embodiments, the synthetic combinations described hereincomprise seeds and the first endophyte is associated with the seeds as acoating on the surface of the seeds; and/or comprises seedlings and thefirst endophyte is contacted with the seedlings as a spray applied toone or more leaves and/or one or more roots of the seedlings; and/orfurther comprises one or more additional endophyte species.

The effective amount of the synthetic combinations described herein canbe, for example, 1×10̂3 CFU or spores/per seed; from about 1×10̂2 CFU orspores/per seed to about 1×10̂8 CFU or spores/per seed.

In some embodiments, the seed is a seed from an agricultural plant. Insome embodiments, the seed is a transgenic seed.

The synthetic combinations described herein can further comprise, e.g.,one or more of the following: a stabilizer, or a preservative, or acarrier, or a surfactant, or an anticomplex agent, or any combinationthereof. In some embodiments, the synthetic combinations describedherein further comprising one or more of the following: fungicide,nematicide, bactericide, insecticide, and herbicide.

Also described herein are a plurality of any of the syntheticcombinations described herein, placed in a medium that promotes plantgrowth, said medium selected from the group consisting of: soil,hydroponic apparatus, and artificial growth medium. In some embodiments,the plurality of synthetic combinations are placed in the soil in rows,with substantially equal spacing between each seed within each row. Alsodescribed herein are a plurality of synthetic combinations confinedwithin an object selected from the group consisting of: bottle, jar,ampule, package, vessel, bag, box, bin, envelope, carton, container,silo, shipping container, truck bed, and case; in some embodiments, thesynthetic combinations are shelf-stable.

Also described herein are plants grown from the synthetic combinationsdescribed herein, said plant exhibiting an improved phenotype ofagronomic interest, selected from the group consisting of: diseaseresistance, drought tolerance, heat tolerance, cold tolerance, salinitytolerance, metal tolerance, herbicide tolerance, chemical tolerance,improved water use efficiency, improved nitrogen utilization, improvednitrogen fixation, pest resistance, herbivore resistance, pathogenresistance, increased yield, increased yield under water-limitedconditions, health enhancement, vigor improvement, growth improvement,photosynthetic capability improvement, nutrition enhancement, alteredprotein content, altered oil content, increased biomass, increased shootlength, increased root length, improved root architecture, increasedseed weight, altered seed carbohydrate composition, altered seed oilcomposition, number of pods, delayed senescence, stay-green, and alteredseed protein composition. In one embodiment, described herein is a plantor progeny of the plant of the synthetic combinations described herein,wherein said plant or progeny of the plant comprises in at least one ofits plant elements said endophytes.

Described herein is an agricultural plant, or portion or tissue thereof,comprising a formulation comprising an endophyte that is common to atleast two donor plant types that is disposed on an exterior surface ofor within the plant in an amount effective to colonize the plant, and inan amount effective to provide a benefit to the modern agriculturalplant. In some embodiments, the endophyte comprises a nucleic acidsequence that is at least 95% identical to a nucleic acid sequenceprovided in Tables 5-14. Also described herein is a modern agriculturalplant, or portion or tissue thereof, comprising a formulation comprisingan endophytic microbial entity derived from an ancestral agriculturalplant that is disposed on an exterior surface of or within the plant inan amount effective to colonize the plant, and in an amount effective toprovide a benefit to the modern agricultural plant. In some embodiments,the endophyte comprises a nucleic acid sequence that is at least 95%identical to a nucleic acid sequence provided in Tables 16-23.

The plants described herein are provided a benefit that is, for example,selected from the group consisting of increased root biomass, increasedroot length, increased height, increased shoot length, increased leafnumber, increased water use efficiency, increased overall biomass,increase grain yield, increased photosynthesis rate, increased toleranceto drought, increased heat tolerance, increased salt tolerance,increased resistance to nematode stress, increased resistance to afungal pathogen, increased resistance to a bacterial pathogen, increasedresistance to a viral pathogen, a detectable modulation in the level ofa metabolite, and a detectable modulation in the proteome relative to areference plant. In some embodiments, at least two benefits are providedto the agricultural plant.

In some embodiments, the plant is contacted with at least 100 CFU orspores, at least 300 CFU or spores, at least 1,000 CFU or spores, atleast 3,000 CFU or spores, at least 10,000 CFU or spores, at least30,000 CFU or spores, at least 100,000 CFU or spores, at least 300,000CFU or spores, at least 1,000,000 CFU or spores or more, of theendophyte.

In some embodiments, the plant is a seed. In some embodiments, the plantis a seed and the population is disposed on the surface of the seed.

The plants described herein are can include at least two endophyticmicrobial entities comprising a nucleic acid sequence that is at least97% identical to any nucleic acid provided in Tables 1-10 in an amounteffective to colonize the mature agricultural plant.

In some embodiments, the plant is a monocot, e.g., selected from thegroup consisting of corn, wheat, barley and rice. In some embodiments,the plant is a dicot, e.g., selected from the group consisting of asoybean, canola, cotton, tomato and pepper.

In some embodiments of the plants described herein, the endophyte can bedisposed in an amount effective to be detectable within a target tissueof the mature target tissue of the mature agricultural plant selectedfrom a fruit, seed, leaf, root or portion thereof

In some embodiments of the plants described herein, the target tissuecan be selected from the group consisting of the root, shoot, leaf,flower, fruit and seed.

In some embodiments of the plants described herein, the population canbe detected in an amount of at least 100 CFU or spores, at least 300 CFUor spores, at least 1,000 CFU or spores, at least 3,000 CFU or spores,at least 10,000 CFU or spores, at least 30,000 CFU or spores, at least100,000 CFU or spores, at least 300,000 CFU or spores, at least1,000,000 CFU or spores, or more, in the plant or target tissue thereof.

In some embodiments of the plants described herein, the population of isdisposed in an amount effective to be detectable in the rhizospheresurrounding the plant. For example, the population can be detected in anamount of at least 100 CFU or spores, at least 300 CFU or spores, atleast 1,000 CFU or spores, at least 3,000 CFU or spores, at least 10,000CFU or spores, at least 30,000 CFU or spores, at least 100,000 CFU orspores, at least 300,000 CFU or spores, at least 1,000,000 CFU orspores, or more, in the rhizosphere surrounding the plant.

In some embodiments of the plants described herein, the population isdisposed in an amount effective to detectably increase the biomass ofthe plant. For example, the biomass of the plant can be detectablyincreased by at least 1%, at least 2%, at least 3%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, or more, when compared with a reference agricultural plant.

In some embodiments of the plants described herein, the population isdisposed in an amount effective to increase the biomass of a fruit orseed of the plant. For example, the biomass of the fruit or seed of theplant can be detectably increased by at least 1%, at least 2%, at least3%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, or more, when compared with the fruit or seedof a reference agricultural plant.

In some embodiments of the plants described herein, the population isdisposed in an amount effective to increase the height of the plant. Forexample, the height of the plant can be detectably increased by at least1%, at least 2%, at least 3%, at least 5%, at least 10%, at least 15%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, or more, whencompared with the height of a reference agricultural plant.

In some embodiments of the plants described herein, the population isdisposed in an amount effective to effective to increase resistance toany of the stress conditions selected from the group consisting of adrought stress, heat stress, cold stress, salt stress, and low mineralstress. For example, the population can be disposed in an amounteffective to effective to increase resistance to any of the bioticstress conditions selected from the group consisting of a nematodestress, insect herbivory stress, fungal pathogen stress, bacterialpathogen stress, and viral pathogen stress.

Also described herein is agricultural product comprising a 1000 seedweight amount of a synthetic compositions described herein. In someembodiments, the endophytes are present in a concentration of from about10̂2 to about 10̂5 CFU or spores/ml or from about 10̂5 to about 10̂8 CFU orspores/seed. In some embodiments, the benefit is selected from the groupconsisting of: increased root biomass, increased root length, increasedheight, increased shoot length, increased leaf number, increased wateruse efficiency, increased tolerance to low nitrogen stress, increasednitrogen use efficiency, increased overall biomass, increase grainyield, increased photosynthesis rate, increased tolerance to drought,increased heat tolerance, increased salt tolerance, increased resistanceto nematode stress, increased resistance to a fungal pathogen, increasedresistance to a bacterial pathogen, increased resistance to a viralpathogen, a detectable modulation in the level of a metabolite, and adetectable modulation in the proteome relative to a reference plant, ora combination thereof.

Described herein are commodity plant products comprising the plantsdescribed herein. In some embodiments, the product is a grain, a flour,a starch, a syrup, a meal, an oil, a film, a packaging, a nutraceuticalproduct, a pulp, an animal feed, a fish fodder, a bulk material forindustrial chemicals, a cereal product, a processed human-food product,a sugar or an alcohol and protein. Also described herein are method ofproducing a commodity plant product, comprising: obtaining a plant orplant tissue from any of the plants described herein, or progeny orderivative thereof, and producing the commodity plant product therefrom.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Non-metric multidimensional scaling plot showing the differencesin overall bacterial community composition between (A) wild and moderncorn seeds and (B) wild and modern wheat seeds. Points representcommunity composition for an individual sample. Points closer togetherrepresent more similar communities while points further apart representmore dissimilar communities.

FIG. 2. Non-metric multidimensional scaling plot showing the differencesin overall fungal community composition between (A) wild and modern cornseeds and (B) wild and modern wheat seeds. Points represent communitycomposition for an individual sample. Points closer together representmore similar communities while points further apart represent moredissimilar communities.

FIG. 3. Differences in seed bacterial diversity among various cultivarsof corn. Shown are the Shannon Diversity indices of bacterialcommunities found in Teosinte, Landrace, Inbred and Modern cultivars ofcorn.

FIG. 4. Differences in seed bacterial diversity among various cultivarsof wheat. Shown are the Shannon Diversity indices of bacterialcommunities found in Wild, Landrace and Modern cultivars of wheat.

FIG. 5. Differences in seed fungal diversity across different cultivarsof corn. Shown are the Shannon Diversity indices of fungal communitiesfound in Teosinte, Landrace, Inbred and Modern cultivars of corn,illustrating the lower diversity of fungal communities within moderncorn.

FIG. 6. Differences in seed fungal diversity across different cultivarsof wheat. Shown are the Shannon Diversity indices of fungal communitiesfound in Wild, Landrace and Modern cultivars of wheat which, as withcorn, demonstrate a reduced diversity of fungal communities withinmodern corn.

DETAILED DESCRIPTION

The inventors have undertaken a systematic comparison of the microbialcommunities that reside within a wide diversity of agricultural plants.The present invention is based on the striking finding that keyconstituents of the plant microbiome can be shared across diverse cropvarieties, and the identification of bacterial and fungal species thatprovide diverse advantages to novel crop hosts via heterologousadministration. As such, the endophytic microbes useful for theinvention generally relate to endophytic microbes that are present inagricultural plants.

Currently, the generally accepted view of plant endophytic communitiesfocuses on their homologous derivation, predominantly from the soilcommunities in which the plants are grown (Hallman, J., et al., (1997)Canadian Journal of Microbiology. 43(10): 895-914). Upon observingtaxonomic overlap between the endophytic and soil microbiota in A.thaliana, it was stated, “Our rigorous definition of an endophyticcompartment microbiome should facilitate controlled dissection ofplant-microbe interactions derived from complex soil communities”(Lundberg et al., (2012) Nature. 488, 86-90). There is strong support inthe art for soil representing the repository from which plant endophytesare derived. New Phytologist (2010) 185: 554-567. Notable plant-microbeinteractions such as mycorrhyzal fungi and bacterial rhizobia fit theparadigm of soil-based colonization of plant hosts and appear toprimarily establish themselves independently of seed. As a result offocusing attention on the derivation of endophytes from the soil inwhich the target agricultural plant is currently growing, there has beenan inability to achieve commercially significant improvements in plantyields and other plant characteristics such as altered oil content,altered protein content, altered seed carbohydrate composition, alteredseed oil composition, and altered seed protein composition, chemicaltolerance, cold tolerance, delayed senescence, disease resistance,drought tolerance, ear weight, growth improvement, health enhancement,heat tolerance, herbicide tolerance, herbivore resistance, improvednitrogen fixation, improved nitrogen utilization, improved rootarchitecture, improved water use efficiency, increased biomass,increased root length, increased seed weight, increased shoot length,increased yield, increased yield under water-limited conditions, kernelmass, kernel moisture content, metal tolerance, number of ears, numberof kernels per ear, number of pods, nutrition enhancement, pathogenresistance, pest resistance, photosynthetic capability improvement,salinity tolerance, stay-green, vigor improvement, increased dry weightof mature seeds, increased fresh weight of mature seeds, increasednumber of mature seeds per plant, increased chlorophyll content,increased number of pods per plant, increased length of pods per plant,reduced number of wilted leaves per plant, reduced number of severelywilted leaves per plant, and increased number of non-wilted leaves perplant, a detectable modulation in the level of a metabolite, adetectable modulation in the level of a transcript, and a detectablemodulation in the proteome relative to a reference plant.

In part, the present invention describes preparations of novel seed- orplant-derived endophytes, and the creation of synthetic combinations ofagricultural seeds and/or seedlings with heterologous seed- orplant-derived endophytes and formulations containing the syntheticcombinations, as well as the recognition that such syntheticcombinations display a diversity of beneficial properties present in theagricultural plants and the associated endophyte populations newlycreated by the present inventors. Such beneficial properties includemetabolism, transcript expression, proteome alterations, morphology, andthe resilience to a variety of environmental stresses, and thecombination of a plurality of such properties.

Little attention has been provided in the art to understand the role ofplant elements as reservoirs for microbes that can efficiently populatethe endosphere of agricultural plants. While the concept that plantelements may harbor plant pathogens was promoted by Baker and Smith(Annu Rev Phytopathol 14: 311-334(1966)), and the understanding thatbacterial and fungal pathogens are known to be able to infect plantelements, the ability to harness endophytes derived from a broadspectrum of plant elements to heterologously confer single or multipleadvantages to agricultural crops was previously unrecognized. As thepresence of detectable pathogens in a plant element lot can necessitatedestruction of vast numbers of agricultural germplasm (Gitaitis, R. andWalcott, R. (2007) Annu Rev. Phytopathol. 45:371-97), safety concernshave surrounded the consideration of seed-associated microbes ornon-soil endophytes. Moreover, when seed pathogens are detected, theirtransfer to the growing plant can be highly inefficient. For example, astudy of seed-based transmission of the seed pathogen, Pantoeastewartii, found that seed produced from a population ofpathogen-infected plants gave rise to infected seedlings in only 0.0029%of cases (1 of 34,924 plants) and artificially infected kernels onlygave rise to infected seedlings in 0.022% of cases (Block, C. C., etal., (1998). Plant disease. 82(7). 775-780). Thus, the efficiency withwhich plants introduce microbes into their seeds, and with whichmicrobes within seeds propagate within the resulting plant tissues, hasbeen previously thought to be low and often substantially variable.Thus, the potential for microbial content within seeds to populate theresulting plant has been unclear.

The potential for agricultural plant elements to serve as reservoirs fornon-pathogenic microbes also remains controversial (Hallman, J., et al.,(1997) Canadian Journal of Microbiology. 43(10): 895-914). Sato, et al.,did not detect any bacteria inside rice seeds ((2003) In. Morishima, H.(ed.) The Natural History of Wild Rice—Evolution Ecology of Crop. p.91-106) and Mundt and Hinkle only obtained endophytes from seed sampleswhere seed coats had been broken or fractured in 29 kinds of plant seed(Appl Environ Microbiol. (1976) 32(5):694-8). Another group detectedsimply bacterial populations inside rice seeds ranging in populationsize from 10̂2 to 10̂6 CFU/g fresh weight (Okunishi, S., et al., (2005)Microbes and Environment. 20:168-177). Rosenblueth et al described seedsto harbor very simple microbial communities with significant variabilityof the microbial communities between individual maize seeds, includingsubstantial variability between seeds taken from the same cobb(Rosenblueth, M. et al, Seed Bacterial Endophytes: Common Genera,Seed-to-Seed Variability and Their Possible Role in Plants; Proc.XXVIIIth IHC—IS on Envtl., Edaphic & Gen. Factors; Affecting Plants,Seeds and Turfgrass; Eds.: G. E. Welbaum et al. Acta Hort. 938, ISHS2012).

These findings demonstrate limitations recognized in the art regardingthe attempted use of endophytes derived from seeds; i.e., maize seedsappear to contain limited taxonomic diversity, and that the microbiotaof individual seeds produced by plants is often distinct, indicatingthat there may not be single seed- or plant-derived symbionts capable ofproviding benefits across a large population of agricultural plants andin specific, the utilization of endophytes on seed. For example,characterization of ˜15 pooled seeds from within various cultivars fromthe genus Zea showed that populations of maize seeds tend to harbor avery limited number of taxa that appear to be conserved across modernand ancestral variants, and that the maize seed content of such taxa islow and substantially variable. It is unclear whether the presence ofsuch limited taxa resulted from common storage conditions, environmentalcontamination, or a potential vertical transmission of microbes viaseeds, and also uncertain was the applicability of such limited taxa inincreasing agricultural yield. Notably, 99% of these strains were shownto provide detrimental or to lack beneficial effects on agriculturalplants, e.g., when tested in a potato growth assay (Johnston-Monje D,Raizada M N (2011) Conservation and Diversity of Seed AssociatedEndophytes in Zea across Boundaries of Evolution, Ethnography andEcology. PLoS ONE 6(6): e20396. doi:10.1371/journal.pone.0020396).Further, some of the microbes isolated bear close evolutionary relationto plant pathogens, making it possible that such microbes represent alatent reservoir of pathogens, rather than potentially beneficialconstituents.

Surprisingly, we discovered here that seed- or plant-derived endophytescan confer significant advantages to agricultural crops, spanning growthunder normal and stressed conditions, altered expression of key planthormones, altered expression of key transcripts in the plant, and otherdesirable features. Provided are novel compositions, methods, andproducts related our invention's ability to overcome the limitations ofthe prior art in order to provide reliable increases in crop yield,biomass, germination, vigor, stress resilience, and other properties toagricultural crops.

Our invention is surprising for multiple reasons based on the previousdemonstrations in the art. Notably, there has been a lack of clarityrelated to whether endophytes are associated with healthy plantelements, whether microbes isolated from plant elements couldefficiently colonize the host if disposed on the exterior of a plantelement or seedling, and whether such microbes would confer a beneficialor detrimental effects on hosts. It has been further unclear whether theheterologous application of such microbes to distinct plant elementsfrom which they were derived could provide beneficial effects.

We find that beneficial microbes from within the conserved microbialtaxa can be robustly derived from agricultural plant elements,optionally cultured, administered heterologously to agricultural plantelements or seedlings, and colonize the resulting plant tissues withhigh efficiency to confer multiple beneficial properties. This issurprising given the variability observed in the art in microbeisolation from healthy plant elements and the previous observations ofinefficient plant element pathogen colonization of plant host's tissues.Further, the ability of heterologously disposed seed- or plant-derivedendophytes to colonize seeds and seedlings from the exterior of seeds issurprising, given that such endophytes can be isolated from withininternal seed tissues and therefore do not natively need the capacity toexternally penetrate and invade into host tissues.

Prior characterization of microbial content of seeds has indicated thatmicrobial concentrations in seeds can be variable and are generally verylow (ie, less than 10, 100, 10̂3, 10̂4, 10̂5 CFUs/seed). As such, it hasbeen unclear whether altered or increased concentrations of microbesassociated with seeds could be beneficial. We find that microbes canconfer beneficial properties across a range of concentrations.

A significant limitation of the existing art in endophytes is the verylimited perspective on endophyte community compositions across adiversity of plant genotypes and environments. This has led to endophyteisolations that have lacked the ability to colonize multiple hosts or toreproducibly confer benefits in multiple locations and soil types.

The inventors conceived that the bacterial and fungal microbiota oflarge numbers of agricultural seeds and wild seeds from a diversity ofgeographic locations would have an improved ability to colonize andbenefit multiple plant genotypes across multiple environments. Theinventors have developed a method to introduce isolated endophytes toanother plant by coating the microbes onto the surface of a seed of aplant. By combining an endophyte sourced from one plant, it is possibleto transfer new beneficial agronomic traits onto an agricultural plant,which therefore holds great promise for increasing agriculturalproductivity. Additionally, as demonstrated herein, the microbialendophytes were in many cases able to additively confer benefits torecipient seeds, seedlings, or plants.

Combining a selected plant species, OTU, strain or cultivar with one ormore types of endophytes thus provides mechanisms by which, alone or inparallel with plant breeding and transgenic technologies, is providedimproved yield from crops and generation of products thereof. Therefore,in one aspect, the present invention provides a synthetic combination ofa seed of a first plant and a preparation of an endophyte that is coatedonto the surface of the seed of the first plant such that the endophyteis present at a higher level on the surface of the seed than is presenton the surface of an uncoated reference seed, wherein the endophyte isisolated from the inside the seed of a second plant. As describedherein, the combination is achieved by artificial coating, application,or other infection of a seed of a plant with an endophyte strain. Insome embodiments, endophytes are introduced onto the surface of hostplant seeds, which upon cultivation confer improved agronomic traits tosaid host plant, which may then generate progeny seeds.

DEFINITIONS

A “synthetic combination” includes a combination of a host plant and anendophyte. The combination may be achieved, for example, by coating thesurface of the seed of a plant, such as an agricultural plant, or hostplant tissues with an endophyte.

As used herein, an “agricultural seed” is a seed used to grow a plant inagriculture (an “agricultural plant”). The seed may be of a monocot ordicot plant, and is planted for the production of an agriculturalproduct, for example grain, food, feed, fiber, fuel, etc. As usedherein, an agricultural seed is a seed that is prepared for planting,for example, in farms for growing.

An “endophyte” or “endophytic entity” or “endophytic microbe” is anorganism capable of living within a plant or is otherwise associatedtherewith, and does not cause disease or harm the plant otherwise.Endophytes can occupy the intracellular or extracellular spaces of planttissue, including the leaves, stems, flowers, fruits, seeds, or roots.An endophyte can be for example a bacterial or fungal organism, and canconfer a beneficial property to the host plant such as an increase inyield, biomass, resistance, or fitness. An endophyte can be a fungus, ora bacterium. As used herein, the term “microbe” is sometimes used todescribe an endophyte. Further, “endophyte” means a microbe (typically afungus or a bacterium) that is associated with a plant tissue and is ina symbiotic or other beneficial relationship with said plant tissue. Asused herein, an “endophytic component” refers to a composition orstructure that is part of the endophyte.

As used herein, the term “bacteria” or “bacterium” refers in general toany prokaryotic organism, and may reference an organism from eitherKingdom Eubacteria (Bacteria), Kingdom Archaebacteria (Archae), or both.

“Internal Transcribed Spacer” (ITS) refers to the spacer DNA (non-codingDNA) situated between the small-subunit ribosomal RNA (rRNA) andlarge-subunit rRNA genes in the chromosome or the correspondingtranscribed region in the polycistronic rRNA precursor transcript.

A “complex network” means a plurality of endophyte entities (e.g.,simple bacteria or simple fungi, complex fungi, or combinations thereof)co-localized in an environment, such as on or within an agriculturalplant. Preferably, a complex network includes two or more types ofendophyte entities that synergistically interact, such synergisticendophytic populations capable of providing a benefit to theagricultural seed, seedling, or plant derived thereby.

A “population” of endophytes refers to the presence of more than oneendophyte in a particular environment. The population may comprise morethan one individual of the same taxonomy or more than one taxonomy ofindividuals. For example, a population may comprise 10̂2 colonies ofCladosporium. In another example, a population may comprise 10̂2 coloniesof Cladosporium and 10̂3 colonies of Penicillium. A population may ingeneral, but not be limited to, comprises individuals that are relatedby some feature, such as being in the same environment at the same time,or by virtue of sharing some phenotype such as ability to metabolize aparticular substrate.

The terms “pathogen” and “pathogenic” in reference to a bacterium orfungus includes any such organism that is capable of causing oraffecting a disease, disorder or condition of a host comprising theorganism.

A “spore” or a population of “spores” refers to bacteria or fungi thatare generally viable, more resistant to environmental influences such asheat and bactericidal or fungicidal agents than other forms of the samebacteria or fungi, and typically capable of germination and out-growth.Bacteria and fungi that are “capable of forming spores” are thosebacteria and fungi comprising the genes and other necessary abilities toproduce spores under suitable environmental conditions.

As used herein, a “colony-forming unit” (“CFU”) is used as a measure ofviable microorganisms in a sample. A CFU is an individual viable cellcapable of forming on a solid medium a visible colony whose individualcells are derived by cell division from one parental cell.

The term “isolated” is intended to specifically reference an organism,cell, tissue, polynucleotide, or polypeptide that is removed from itsoriginal source and purified from additional components with which itwas originally associated. For example, an endophyte may be consideredisolated from a seed if it is removed from that seed source and purifiedso that it is isolated from any additional components with which it wasoriginally associated. Similarly, an endophyte may be removed andpurified from a plant or plant element so that it is isolated and nolonger associated with its source plant or plant element.

A “plant element” is intended to generically reference either a wholeplant or a plant component, including but not limited to plant tissues,parts, and cell types. A plant element is preferably one of thefollowing: whole plant, seedling, meristematic tissue, ground tissue,vascular tissue, dermal tissue, seed, leaf, root, shoot, stem, flower,fruit, stolon, bulb, tuber, corm, kelkis, shoot, bud. As used herein, a“plant element” is synonymous to a “portion” of a plant, and refers toany part of the plant, and can include distinct tissues and/or organs,and may be used interchangeably with the term “tissue” throughout.

Similarly, a “plant reproductive element” is intended to genericallyreference any part of a plant that is able to initiate other plants viaeither sexual or asexual reproduction of that plant, for example but notlimited to: seed, seedling, root, shoot, stolon, bulb, tuber, corm,keikis, or bud.

A “population” of plants, as used herein, can refer to a plurality ofplants that were subjected to the same inoculation methods describedherein, or a plurality of plants that are progeny of a plant or group ofplants that were subjected to the inoculation methods. In addition, apopulation of plants can be a group of plants that are grown from coatedseeds. The plants within a population will typically be of the samespecies, and will also typically share a common genetic derivation.

As used herein, an “agricultural seed” is a seed used to grow a planttypically used in agriculture (an “agricultural plant”). The seed may beof a monocot or dicot plant, and may be planted for the production of anagricultural product, for example feed, food, fiber, fuel, etc. As usedherein, an agricultural seed is a seed that is prepared for planting,for example, in farms for growing.

“Agricultural plants”, or “plants of agronomic importance”, includeplants that are cultivated by humans for food, feed, fiber, and fuelpurposes. Agricultural plants include monocotyledonous species such as:maize (Zea mays), common wheat (Triticum aestivum), spelt (Triticumspelta), einkorn wheat (Triticum monococcum), emmer wheat (Triticumdicoccum), durum wheat (Triticum durum), Asian rice (Oryza sativa),African rice (Oryza glabaerreima), wild rice (Zizania aquatica, Zizanialatifolia, Zizania palustris, Zizania texana), barley (Hordeum vulgare),Sorghum (Sorghum bicolor), Finger millet (Eleusine coracana), Prosomillet (Panicum miliaceum), Pearl millet (Pennisetum glaucum), Foxtailmillet (Setaria italica), Oat (Avena sativa), Triticale (Triticosecale),rye (Secale cereal), Russian wild rye (Psathyrostachys juncea), bamboo(Bambuseae), or sugarcane (e.g., Saccharum arundinaceum, Saccharumbarberi, Saccharum bengalense, Saccharum edule, Saccharum munja,Saccharum officinarum, Saccharum procerum, Saccharum ravennae, Saccharumrobustum, Saccharum sinense, or Saccharum spontaneum); as well asdicotyledonous species such as: soybean (Glycine max), canola andrapeseed cultivars (Brassica napus), cotton (genus Gossypium), alfalfa(Medicago sativa), cassava (genus Manihot), potato (Solanum tuberosum),tomato (Solanum lycopersicum), pea (Pisum sativum), chick pea (Cicerarietinum), lentil (Lens culinaris), flax (Linum usitatissimum) and manyvarieties of vegetables.

A “host plant” includes any plant, particularly a plant of agronomicimportance, which an endophytic entity such as an endophyte cancolonize. As used herein, an endophyte is said to “colonize” a plant orseed when it can be stably detected within the plant or seed over aperiod time, such as one or more days, weeks, months or years, in otherwords, a colonizing entity is not transiently associated with the plantor seed. Such host plants are preferably plants of agronomic importance.It is contemplated that any element, or more than one element, of thehost plant may be colonized with an endophyte to thus confer a hoststatus to the plant. The initial inoculated element may additionally bedifferent than the element to which the endophyte localizes. Anendophyte may localize to different elements of the same plant in aspatial or temporal manner. For example, a seed may be inoculated withan endophyte, and upon germination, the endophyte may localize to roottissue.

A “non-host target” means an organism or chemical compound that isaltered in some way after contacting a host plant or host fungus thatcomprises an endophyte, as a result of a property conferred to the hostplant or host fungus by the endophyte.

As used herein, an “ancestral” variety of a plant refers generally to avariety or species of a plant that is either a wild ancestor orundomesticated species of agricultural plants. Such ancestral varietiesare generally distinguished from agricultural plants used in large-scaleagricultural practices in use today in that the ancestral varieties werenot extensively bred, and are generally open-pollinated. As used herein,ancestral varieties include landrace varieties, heirloom varieties, andprogenitor species.

A “modern” variety of a plant refers to a non-ancestral variety of aplant.

As used herein, a “hybrid plant” refers generally refers to a plant thatis the product of a cross between two genetically different parentalplants. A hybrid plant is generated by either a natural or artificialprocess of hybridization whereby the entire genome of one species,variety cultivar, breeding line or individual plant is combined intra-or interspecifically into the genome of species, variety or cultivar orline, breeding line or individual plant by crossing.

An “inbred plant”, as used herein, refers to a plant or plant line thathas been repeatedly crossed or inbred to achieve a high degree ofgenetic uniformity, and low heterozygosity, as is known in the art.

The term “isoline” is a comparative term, and references organisms thatare genetically identical, but may differ in treatment. In one example,two genetically identical maize plant embryos may be separated into twodifferent groups, one receiving a treatment (such as transformation witha heterologous polynucleotide, to create a genetically modified plant)and one control that does not receive such treatment. Any phenotypicdifferences between the two groups may thus be attributed solely to thetreatment and not to any inherency of the plant's genetic makeup. Inanother example, two genetically identical seeds may be treated with aformulation that introduces an endophyte composition. Any phenotypicdifferences between the plants grown from those seeds may be attributedto the treatment, thus forming an isoline comparison.

Similarly, by the term “reference agricultural plant”, it is meant anagricultural plant of the same species, strain, or cultivar to which atreatment, formulation, composition or endophyte preparation asdescribed herein is not administered/contacted. A reference agriculturalplant, therefore, is identical to the treated plant with the exceptionof the presence of the endophyte and can serve as a control fordetecting the effects of the endophyte that is conferred to the plant.

A “reference environment” refers to the environment, treatment orcondition of the plant in which a measurement is made. For example,production of a compound in a plant associated with an endophyte can bemeasured in a reference environment of drought stress, and compared withthe levels of the compound in a reference agricultural plant under thesame conditions of drought stress. Alternatively, the levels of acompound in plant associated with an endophyte and referenceagricultural plant can be measured under identical conditions of nostress.

In some embodiments, the invention contemplates the use of microbes thatare “exogenous” to a seed or plant. As used herein, a microbe isconsidered exogenous to the seed or plant if the seed or seedling thatis unmodified (e.g., a seed or seedling that is not treated with theendophytic microbial population descried herein) does not contain themicrobe.

In some embodiments, a microbe can be “endogenous” to a seed or plant.As used herein, a microbe is considered “endogenous” to a plant or seed,if the endophyte or endophyte component is derived from, or is otherwisefound in, a plant element of the plant specimen from which it issourced. In embodiments in which an endogenous endophyte is applied, theendogenous microbe is applied in an amount that differs from the levelstypically found in the plant.

In some embodiments, the invention uses endophytes that are heterologousto a plant element, for example in making synthetic combinations oragricultural formulations. A microbe is considered heterologous to theseed or plant if the seed or seedling that is unmodified (e.g., a seedor seedling that is not treated with an endophyte population describedherein) does not contain detectable levels of the microbe. For example,the invention contemplates the synthetic combinations of seeds orseedlings of agricultural plants and an endophytic microbe population(e.g., an isolated bacterium), in which the microbe population is“heterologously disposed” on the exterior surface of or within a tissueof the agricultural seed or seedling in an amount effective to colonizethe plant. A microbe is considered “heterologously disposed” on thesurface or within a plant (or tissue) when the microbe is applied ordisposed on the plant in a number that is not found on that plant beforeapplication of the microbe. For example, an endophyte population that isdisposed on an exterior surface or within the seed can be an endophyticbacterium that may be associated with the mature plant, but is not foundon the surface of or within the seed. As such, a microbe is deemedheterologously disposed when applied on the plant that either does notnaturally have the microbe on its surface or within the particulartissue to which the microbe is disposed, or does not naturally have themicrobe on its surface or within the particular tissue in the numberthat is being applied. The term “exogenous” can be used interchangeablywith “heterologous.” For example, a fungal endophyte that is normallyassociated with leaf tissue of a cupressaceous tree sample would beconsidered heterologous to leaf tissue of a maize plant. In anotherexample, an endophyte that is normally associated with leaf tissue of amaize plant is considered heterologous to a leaf tissue of another maizeplant that naturally lacks said endophyte. In another example, a fungalendophyte that is normally associated at low levels in a plant isconsidered heterologous to that plant if a higher concentration of thatendophyte is introduced into the plant. In another example, an endophytethat is comprised within one fungus would be considered heterologous ifplaced in a different fungus. In yet another example, an endophyte thatis associated with a tropical grass species would be consideredheterologous to a wheat plant.

For the avoidance of doubt, “heterologously disposed” contemplates useof microbes that are “exogenous” to a seed or plant.

In some cases, the present invention contemplates the use of microbesthat are “compatible” with agricultural chemicals, for example, afungicide, an anti-bacterial compound, or any other agent widely used inagricultural which has the effect of interfering with optimal growth ofmicrobes. As used herein, a microbe is “compatible” with an agriculturalchemical, when the microbe is modified or otherwise adapted to grow in,or otherwise survive, the concentration of the agricultural chemicalused in agriculture. For example, a microbe disposed on the surface of aseed is compatible with the fungicide metalaxyl if it is able to survivethe concentrations that are applied on the seed surface.

“Biomass” means the total mass or weight (fresh or dry), at a giventime, of a plant tissue, plant tissues, an entire plant, or populationof plants, usually given as weight per unit area. The term may alsorefer to all the plants or species in the community (community biomass).

Some of the compositions and methods described herein involve endophyticmicrobes in an amount effective to colonize a plant. As used herein, amicrobe is said to “colonize” a plant or seed when it can exist in anendophytic relationship with the plant in the plant environment, forexample inside the plant or a part or tissue thereof, including theseed.

The compositions and methods herein may provide for an improved“agronomic trait” or “trait of agronomic importance” to a host plant,which may include, but not be limited to, the following: altered oilcontent, altered protein content, altered seed carbohydrate composition,altered seed oil composition, and altered seed protein composition,chemical tolerance, cold tolerance, delayed senescence, diseaseresistance, drought tolerance, ear weight, growth improvement, healthenhancement, heat tolerance, herbicide tolerance, herbivore resistance,improved nitrogen fixation, improved nitrogen utilization, improved rootarchitecture, improved water use efficiency, increased biomass,increased root length, increased seed weight, increased shoot length,increased yield, increased yield under water-limited conditions, kernelmass, kernel moisture content, metal tolerance, number of ears, numberof kernels per ear, number of pods, nutrition enhancement, pathogenresistance, pest resistance, photosynthetic capability improvement,salinity tolerance, stay-green, vigor improvement, increased dry weightof mature seeds, increased fresh weight of mature seeds, increasednumber of mature seeds per plant, increased chlorophyll content,increased number of pods per plant, increased length of pods per plant,reduced number of wilted leaves per plant, reduced number of severelywilted leaves per plant, and increased number of non-wilted leaves perplant, a detectable modulation in the level of a metabolite, adetectable modulation in the level of a transcript, and a detectablemodulation in the proteome, compared to an isoline plant grown from aseed without said seed treatment formulation.

As used herein, the terms “water-limited condition” and “droughtcondition”, or “water-limited” and “drought”, may be usedinterchangeably. For example, a method or composition for improving aplant's ability to grown under drought conditions means the same as theability to grow under water-limited conditions. In such cases, the plantcan be further said to display improved drought tolerance.

Additionally, “altered metabolic function” or “altered enzymaticfunction” may include, but not be limited to, the following: alteredproduction of an auxin, altered nitrogen fixation, altered production ofan antimicrobial compound, altered production of a siderophore, alteredmineral phosphate solubilization, altered production of a cellulase,altered production of a chitinase, altered production of a xylanase,altered production of acetoin.

An “increased yield” can refer to any increase in biomass or seed orfruit weight, seed size, seed number per plant, seed number per unitarea, bushels per acre, tons per acre, kilo per hectare, or carbohydrateyield. Typically, the particular characteristic is designated whenreferring to increased yield, e.g., increased grain yield or increasedseed size.

“Agronomic trait potential” is intended to mean a capability of a plantelement for exhibiting a phenotype, preferably an improved agronomictrait, at some point during its life cycle, or conveying said phenotypeto another plant element with which it is associated in the same plant.For example, a seed may comprise an endophyte that will provide benefitto leaf tissue of a plant from which the seed is grown; in such case,the seed comprising such endophyte has the agronomic trait potential fora particular phenotype (for example, increased biomass in the plant)even if the seed itself does not display said phenotype.

By the term “capable of metabolizing” a particular carbon substrate, itis meant that the endophyte is able to utilize that carbon substrate asan energy source.

The term “synthetic combination” means a plurality of elementsassociated by human endeavor, in which said association is not found innature. In the present invention, “synthetic combination” is used torefer to a treatment formulation associated with a plant element.

A “treatment formulation” refers to a mixture of chemicals thatfacilitate the stability, storage, and/or application of the endophytecomposition(s). In some embodiments, an agriculturally compatiblecarrier can be used to formulate an agricultural formulation or othercomposition that includes a purified endophyte preparation. As usedherein an “agriculturally compatible carrier” refers to any material,other than water, that can be added to a plant element without causingor having an adverse effect on the plant element (e.g., reducing seedgermination) or the plant that grows from the plant element, or thelike.

In some cases, the present invention contemplates the use ofcompositions that are “compatible” with agricultural chemicals, forexample, a fungicide, an anti-complex compound, or any other agentwidely used in agricultural which has the effect of killing or otherwiseinterfering with optimal growth of another organism. As used herein, acomposition is “compatible” with an agricultural chemical when theorganism is modified, such as by genetic modification, e.g., contains atransgene that confers resistance to an herbicide, or is adapted to growin, or otherwise survive, the concentration of the agricultural chemicalused in agriculture. For example, an endophyte disposed on the surfaceof a seed is compatible with the fungicide metalaxyl if it is able tosurvive the concentrations that are applied on the seed surface.

Some compositions described herein contemplate the use of anagriculturally compatible carrier. As used herein an “agriculturallycompatible carrier” is intended to refer to any material, other thanwater, which can be added to a seed or a seedling without causing/havingan adverse effect on the seed, the plant that grows from the seed, seedgermination, or the like.

A “transgenic plant” includes a plant or progeny plant of any subsequentgeneration derived therefrom, wherein the DNA of the plant or progenythereof contains an introduced exogenous DNA segment not naturallypresent in a non-transgenic plant of the same strain. The transgenicplant may additionally contain sequences that are native to the plantbeing transformed, but wherein the “exogenous” gene has been altered inorder to alter the level or pattern of expression of the gene, forexample, by use of one or more heterologous regulatory or otherelements.

As used herein, a nucleic acid has “homology” or is “homologous” to asecond nucleic acid if the nucleic acid sequence has a similar sequenceto the second nucleic acid sequence. The terms “identity”, “percentsequence identity” or “identical” in the context of nucleic acidsequences refer to the residues in the two sequences that are the samewhen aligned for maximum correspondence. There are a number of differentalgorithms known in the art that can be used to measure nucleotidesequence identity. For instance, polynucleotide sequences can becompared using FASTA, Gap or Bestfit, which are programs in WisconsinPackage Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTAprovides alignments and percent sequence identity of the regions of thebest overlap between the query and search sequences. Pearson, MethodsEnzymol. 183:63-98 (1990). The term “substantial homology” or“substantial similarity,” when referring to a nucleic acid or fragmentthereof, indicates that, when optimally aligned with appropriatenucleotide insertions or deletions with another nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 76%, 80%, 85%, or at least about 90%, or at least about 95%, 96%,97%, 98% or 99% of the nucleotide bases, as measured by any well-knownalgorithm of sequence identity, such as FASTA, BLAST or Gap, asdiscussed above.

As used herein, the terms “operational taxonomic unit,” “OTU,” “taxon,”“hierarchical cluster,” and “cluster” are used interchangeably. Anoperational taxon unit (OTU) refers to a group of one or more organismsthat comprises a node in a clustering tree. The level of a cluster isdetermined by its hierarchical order. In one embodiment, an OTU is agroup tentatively assumed to be a valid taxon for purposes ofphylogenetic analysis. In another embodiment, an OTU is any of theextant taxonomic units under study. In yet another embodiment, an OTU isgiven a name and a rank. For example, an OTU can represent a domain, asub-domain, a kingdom, a sub-kingdom, a phylum, a sub-phylum, a class, asub-class, an order, a sub-order, a family, a subfamily, a genus, asubgenus, or a species. In some embodiments, OTUs can represent one ormore organisms from the kingdoms eubacteria, protista, or fungi at anylevel of a hierarchal order. In some embodiments, an OTU represents aprokaryotic or fungal order.

The terms “decreased”, “fewer”, “slower” and “increased” “faster”“enhanced” “greater” as used herein refers to a decrease or increase ina characteristic of the endophyte treated seed or resulting plantcompared to an untreated seed or resulting plant. For example, adecrease in a characteristic may be at least 1%, at least 2%, at least3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least about 60%, at least 75%, at least about 80%, atleast about 90%, at least 100%, at least 200%, at least about 300%, atleast about 400% or more lower than the untreated control and anincrease may be at least 1%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast about 60%, at least 75%, at least about 80%, at least about 90%,at least 100%, at least 200%, at least about 300%, at least about 400%or more higher than the untreated control.

The present invention is directed to methods and compositions ofendophytes, and plant-endophyte combinations that confer a agronomicbenefit in agricultural plants.

Endophytic Microbes

In part, the present invention describes preparations of novel seed- orplant-derived endophytes, including those that are conserved acrossdiverse species and/or cultivars of agricultural plants, and thecreation of synthetic combinations of agricultural seeds and/orseedlings with heterologous seed- or plant-derived endophytes andformulations containing the synthetic combinations, as well as therecognition that such synthetic combinations display a diversity ofbeneficial properties present in the agricultural plants and theassociated endophyte populations newly created by the present inventors.Such beneficial properties include metabolism, transcript expression,proteome alterations, morphology, and the resilience to a variety ofenvironmental stresses, and the combination of a plurality of suchproperties.

In a second aspect, the inventors have undertaken a systematiccomparison of the microbial communities that reside within ancestral andclosely related modern varieties of agricultural plants. The presentinvention is based on the striking differences the microbial compositionbetween the ancestral and modern varieties, and the identification ofbacterial and fungal species that are absent or vastly underrepresentedin modern varieties. As such, the endophytic microbes useful for theinvention generally relate to endophytic microbes that are present inancestral varieties of plants.

Currently, the generally accepted view of plant endophytic communitiesfocuses on their homologous derivation, predominantly from the soilcommunities in which the plants are grown (Hallman, J., et al., (1997)Canadian Journal of Microbiology. 43(10): 895-914). Upon observingtaxonomic overlap between the endophytic and soil microbiota in A.thaliana, it was stated, “Our rigorous definition of an endophyticcompartment microbiome should facilitate controlled dissection ofplant-microbe interactions derived from complex soil communities”(Lundberg et al., (2012) Nature. 488, 86-90). There is strong support inthe art for soil representing the repository from which plant endophytesare derived. New Phytologist (2010) 185: 554-567. Notable plant-microbeinteractions such as mycorrhyzal fungi and bacterial rhizobia fit theparadigm of soil-based colonization of plant hosts and appear toprimarily establish themselves independently of seed. As a result offocusing attention on the derivation of endophytes from the soil inwhich the target agricultural plant is currently growing, there has beenan inability to achieve commercially significant improvements in plantyields and other plant characteristics such as: altered oil content,altered protein content, altered seed carbohydrate composition, alteredseed oil composition, and altered seed protein composition, chemicaltolerance, cold tolerance, delayed senescence, disease resistance,drought tolerance, ear weight, growth improvement, health enhancement,heat tolerance, herbicide tolerance, herbivore resistance, improvednitrogen fixation, improved nitrogen utilization, improved rootarchitecture, improved water use efficiency, increased biomass,increased root length, increased seed weight, increased shoot length,increased yield, increased yield under water-limited conditions, kernelmass, kernel moisture content, metal tolerance, number of ears, numberof kernels per ear, number of pods, nutrition enhancement, pathogenresistance, pest resistance, photosynthetic capability improvement,salinity tolerance, stay-green, vigor improvement, increased dry weightof mature seeds, increased fresh weight of mature seeds, increasednumber of mature seeds per plant, increased chlorophyll content,increased number of pods per plant, increased length of pods per plant,reduced number of wilted leaves per plant, reduced number of severelywilted leaves per plant, and increased number of non-wilted leaves perplant, a detectable modulation in the level of a metabolite, adetectable modulation in the level of a transcript, or a detectablemodulation in the proteome relative to a reference plant.

Little attention has been provided in the art to understand the role ofseeds as reservoirs for microbes that can efficiently populate theendosphere of agricultural plants. While the concept that seeds mayharbor plant pathogens was promoted by Baker and Smith (Annu RevPhytopathol 14: 311-334(1966)), and the understanding that bacterial andfungal pathogens are known to be able to infect seed, the ability toharness endophytes derived from a broad spectrum of seeds toheterologously confer single or multiple advantages to agriculturalcrops was previously unrecognized. As the presence of detectablepathogens in a seed lot can necessitate destruction of vast numbers ofagricultural germplasm (Gitaitis, R. and Walcott, R. (2007) Annu Rev.Phytopathol. 45:371-97), safety concerns have surrounded theconsideration of seed-associated microbes or non-soil endophytes.Moreover, when seed pathogens are detected, their transfer to thegrowing plant can be highly inefficient. For example, a study ofseed-based transmission of the seed pathogen, Pantoea stewartii, foundthat seed produced from a population of pathogen-infected plants gaverise to infected seedlings in only 0.0029% of cases (1 of 34,924 plants)and artificially infected kernels only gave rise to infected seedlingsin 0.022% of cases (Block, C. C., et al., (1998). Plant disease. 82(7).775-780). Thus, the efficiency with which plants introduce microbes intotheir seeds, and with which microbes within seeds propagate within theresulting plant tissues, has been previously thought to be low and oftensubstantially variable. Thus, the potential for microbial content withinseeds to populate the resulting plant has been unclear.

The potential for agricultural seeds to serve as reservoirs fornon-pathogenic microbes also remains controversial (Hallman, J., et al.,(1997) Canadian Journal of Microbiology. 43(10): 895-914). Sato, et al.,did not detect any bacteria inside rice seeds ((2003) In. Morishima, H.(ed.) The Natural History of Wild Rice—Evolution Ecology of Crop. p.91-106) and Mundt and Hinkle only obtained endophytes from seed sampleswhere seed coats had been broken or fractured in 29 kinds of plant seed(Appl Environ Microbiol. (1976) 32(5):694-8). Another group detectedsimply bacterial populations inside rice seeds ranging in populationsize from 10̂2 to 10̂6 CFU/g fresh weight (Okunishi, S., et al., (2005)Microbes and Environment. 20:168-177). Rosenblueth et al described seedsto harbor very simple microbial communities with significant variabilityof the microbial communities between individual maize seeds, includingsubstantial variability between seeds taken from the same cob(Rosenblueth, M. et al, Seed Bacterial Endophytes: Common Genera,Seed-to-Seed Variability and Their Possible Role in Plants; Proc.XXVIIIth IHC—IS on Envtl., Edaphic & Gen. Factors; Affecting Plants,Seeds and Turf; Eds.: G. E. Welbaum et al. Acta Hort. 938, ISHS 2012).

These findings demonstrate limitations recognized in the art regardingthe attempted use of endophytes derived from seeds; i.e., maize seedsappear to contain limited taxonomic diversity, and that the microbiotaof individual seeds produced by plants is often distinct, indicatingthat there may not be single seed- or plant-derived symbionts capable ofproviding benefits across a large population of agricultural plants andin specific, the utilization of endophytes on seed. For example,characterization of ˜15 pooled seeds from within various cultivars fromthe genus Zea showed that populations of maize seeds tend to harbor avery limited number of taxa that appear to be conserved across modernand ancestral variants, and that the maize seed content of such taxa islow and substantially variable. It is unclear whether the presence ofsuch limited taxa resulted from common storage conditions, environmentalcontamination, or a potential vertical transmission of microbes viaseeds, and also uncertain was the applicability of such limited taxa inincreasing agricultural yield. Notably, 99% of these strains were shownto provide detrimental or to lack beneficial effects on agriculturalplants, e.g., when tested in a potato growth assay (Johnston-Monje D,Raizada M N (2011) Conservation and Diversity of Seed AssociatedEndophytes in Zea across Boundaries of Evolution, Ethnography andEcology. PLoS ONE 6(6): e20396. doi:10.1371/journal.pone.0020396).Further, some of the microbes isolated bear close evolutionary relationto plant pathogens, making it possible that such microbes represent alatent reservoir of pathogens, rather than potentially beneficialconstituents.

We discovered here that seed- or plant-derived endophytes can confersignificant advantages to agricultural crops, spanning growth undernormal and stressed conditions, altered expression of key planthormones, altered expression of key transcripts in the plant, and otherdesirable features. Provided are novel compositions, methods, andproducts related our invention's ability to overcome the limitations ofthe prior art in order to provide reliable increases in crop yield,biomass, germination, vigor, stress resilience, and other properties toagricultural crops.

Our invention is surprising for multiple reasons based on the previousdemonstrations in the art. Notably, there is a lack of clarity relatedto whether endophytes are associated with healthy seeds, whethermicrobes isolated from seeds could efficiently colonize the host ifdisposed on the exterior of a seed or seedling, and whether suchmicrobes would confer a beneficial or detrimental effects on hosts. Itis further unclear whether the heterologous application of such microbesto distinct seeds from which they were derived could provide beneficialeffects.

We find that beneficial microbes can be robustly derived fromagricultural seeds, optionally cultured, administered heterologously toagricultural seeds or seedlings, and colonize the resulting planttissues with high efficiency to confer multiple beneficial properties.This is surprising given the variability observed in the art in microbeisolation from healthy seeds and the previous observations ofinefficient seed pathogen colonization of plant host's tissues. Further,the ability of heterologously disposed seed- or plant-derived endophytesto colonize seeds and seedlings from the exterior of seeds issurprising, given that such endophytes can be isolated from withininternal seed tissues and therefore do not natively need the capacity toexternally penetrate and invade into host tissues.

Prior characterization of microbial content of seeds has indicated thatmicrobial concentrations in seeds can be variable and are generally verylow (ie, less than 10, 100, 10̂3, 10̂4, 10̂5 CFUs/seed). As such, it isunclear whether altered or increased concentrations of microbesassociated with seeds could be beneficial. We find that microbes canconfer beneficial properties across a range of concentrations.

We find that seed- or plant-derived endophytes can be heterologouslydisposed onto seedlings of a distinct cultivar, species, or crop typeand confer benefits to those new recipients. For example, seed- orplant-derived endophytes from corn cultivars are heterologously providedto wheat cultivars to confer a benefit. This is surprising given theobservations of distinct microbiome preferences in distinct plant andmammalian hosts and, in particular, the likelihood that microbes derivedfrom seeds have been co-evolved to be specialized to a particular host.

As used herein, endophytes can be isolated from seeds of many distinctplants. In one embodiment, the endophyte can be isolated from the seedof the same crop, and can be from the same cultivar or variety as theseed onto which it is to be coated. For example, seed endophytes from aparticular corn variety can be isolated and coated onto the surface of acorn seed of the same variety. In one particular embodiment, the seed ofthe first plant that is to be coated with the endophyte can comprise adetectable amount of the same endophyte in the interior of the seed. Inanother embodiment, the seed of the first plant that is to be coatedwith the endophyte can comprise a detectable amount of the sameendophyte in the exterior of the seed. For example, an uncoatedreference seed may contain a detectable amount of the same endophytewithin its seed. In yet another embodiment, the endophyte to be coatedonto the seed of the plant is a microbe that is detectably present inthe interior and exterior of the seed from which the endophyte isderived.

In another embodiment, the endophyte can be isolated from a relatedspecies (e.g., an endophyte isolated from Triticum monococcum (einkornwheat) can be coated onto the surface of a T. aestivum (common wheat)seed; or, an endophyte from Hordeum vulgare (barley) can be isolated andcoated onto the seed of another member of the Triticeae family, forexample, seeds of the rye plant, Secale cereale). In still anotherembodiment, the endophyte can be isolated from the seed of a plant thatis distantly related to the seed onto which the endophyte is to becoated. For example, a tomato-derived endophyte is isolated and coatedonto a rice seed.

In one embodiment, the endophyte is an endophytic microbe that wasisolated from a different plant than the inoculated plant. For example,in one embodiment, the endophyte can be an endophyte isolated from adifferent plant of the same species as the inoculated plant. In somecases, the endophyte can be isolated from a species related to theinoculated plant.

We further find that combinations of heterologously disposed seed- orplant-derived endophytes confer additive advantages to plants, includingmultiple functional properties and resulting in seed, seedling, andplant hosts that display single or multiple improved agronomicproperties.

In another embodiment, the endophytic microbe is absent in a seed of amodern variety of a plant. In still another embodiment, the endophyticmicrobe is detectably underrepresented in the seed of a modern varietyof a plant when compared with a related ancestral variety.

According to the present invention, the endophytic microbe can be abacterium. In some embodiments of the present invention, the endophyteis a member of one of the following taxa: Achromobacter,Acidithiobacillus, Acidovorax, Acidovoraz, Acinetobacter, Actinoplanes,Adlercreutzia, Aerococcus, Aeromonas, Afipia, Agromyces, Ancylobacter,Arthrobacter, Atopostipes, Azospirillum, Bacillus, Bdellovibrio,Beijerinckia, Bosea, Bradyrhizobium, Brevibacillus, Brevundimonas,Burkholderia, Candidatus Haloredivivus, Caulobacter, Cellulomonas,Cellvibrio, Chryseobacterium, Citrobacter, Clostridium,Coraliomargarita, Corynebacterium, Cupriavidus, Curtobacterium,Curvibacter, Deinococcus, Delftia, Desemzia, Devosia, Dokdonella,Dyella, Enhydrobacter, Enterobacter, Enterococcus, Erwinia, Escherichia,Escherichia/Shigella, Exiguobacterium, Ferroglobus, Filimonas,Finegoldia, Flavisolibacter, Flavobacterium, Frigoribacterium,Gluconacetobacter, Hafnia, Halobaculum, Halomonas, Halosimplex,Herbaspirillum, Hymenobacter, Klebsiella, Kocuria, Kosakonia,Lactobacillus, Leclercia, Lentzea, Luteibacter, Luteimonas, Massilia,Mesorhizobium, Methylobacterium, Microbacterium, Micrococcus,Microvirga, Mycobacterium, Neisseria, Nocardia, Oceanibaculum,Ochrobactrum, Okibacterium, Oligotropha, Oryzihumus, Oxalophagus,Paenibacillus, Panteoa, Pantoea, Pelomonas, Perlucidibaca, Plantibacter,Polynucleobacter, Propionibacterium, Propioniciclava, Pseudoclavibacter,Pseudomonas, Pseudonocardia, Pseudoxanthomonas, Psychrobacter,Ralstonia, Rheinheimera, Rhizobium, Rhodococcus, Rhodopseudomonas,Roseateles, Ruminococcus, Sebaldella, Sediminibacillus,Sediminibacterium, Serratia, Shigella, Shinella, Sinorhizobium,Sinosporangium, Sphingobacterium, Sphingomonas, Sphingopyxis,Sphingosinicella, Staphylococcus, Stenotrophomonas, Strenotrophomonas,Streptococcus, Streptomyces, Stygiolobus, Sulfurisphaera, Tatumella,Tepidimonas, Thermomonas, Thiobacillus, Variovorax,WPS-2_genera_incertae_sedis, Xanthomonas, Zimmermannella.

In one embodiment, the endophytic bacterium is of a family selected fromthe group consisting of: Bacillaceae, Burkholderiaceae, Comamonadaceae,Enterobacteriaceae, Flavobacteriaceae, Methylobacteriaceae,Microbacteriaceae, Paenibacillileae, Pseudomonnaceae, Rhizobiaceae,Sphingomonadaceae, Xanthomonadaceae.

In one embodiment, the endophytic bacterium is of a genus selected fromthe group consisting of: Acidovorax, Agrobacterium, Bacillus,Burkholderia, Chryseobacterium, Curtobacterium, Enterobacter,Escherichia, Methylobacterium, Paenibacillus, Pantoea, Pseudomonas,Ralstonia, Saccharibacillus, Sphingomonas, and Stenotrophomonas.

In one embodiment, the endophytic bacterium comprising in its genome anucleic acid sequence selected from the group consisting of: SEQ ID NOs:3588, 3589, 3590, 3591, 3592, 3593, 3594, 3595, 3596, 3598, 3599, 3600,3601, 3603, 3604, 3606, 3607, 3608, 3609, 3619, 3620, 3621, 3622, 3623,3624, 3625, 3626, 3627, 3628, 3629, 3630, 3631, 3632, 3633, 3634, 3635,3636, 3637, 3638, 3639, 3641, 3645, 3646, 3648, 3649, 3651, 3652, 3653,3656, 3663, 3664, 3665, 3666, 3667, 3668, 3669, 3670, 3671.

In one embodiment, the endophytic bacterium comprises in its genome anucleic acid sequence that is at least 90% identical, at least 91%identical, at least 92% identical, at least 93% identical, at least 94%identical, at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, at least 99% identical, at least99.5% identical or 100% identical to a sequence selected from the groupconsisting of: SEQ ID NOs: 3588, 3589, 3590, 3591, 3592, 3593, 3594,3595, 3596, 3598, 3599, 3600, 3601, 3603, 3604, 3606, 3607, 3608, 3609,3619, 3620, 3621, 3622, 3623, 3624, 3625, 3626, 3627, 3628, 3629, 3630,3631, 3632, 3633, 3634, 3635, 3636, 3637, 3638, 3639, 3641, 3645, 3646,3648, 3649, 3651, 3652, 3653, 3656, 3663, 3664, 3665, 3666, 3667, 3668,3669, 3670, 3671.

In another embodiment, the endophytic microbe is a fungus. In someembodiments of the present invention, the endophyte is a member of onethe following taxa: Acidomyces acidophilus, Acremonium alternatum,Acremonium pteridii, Acremonium strictum, Acrodictys elaeidicola,Acrostalagmus luteoalbus, Albatrellus higanensis, Albonectriarigidiuscula, Alternaria alternate, Alternaria arborescens, Alternariaconjuncta, Alternaria helianthi, Alternaria longipes, Alternariamalorum, Alternaria metachromatica, Alternaria oregonensis, Alternariaphotistica, Alternaria protenta, Alternaria tenuissima, Alternariatriticina, Alternaria zinniae, Amorphotheca resinae, Ampelomyces humuli,Anthostomella protege, Apiognomonia errabunda, Aposphaeria populina,Arthrinium sacchari, Aspergillus aculeatus, Aspergillus niger,Aspergillus versicolor, Athelia bombacina, Aureobasidium pullulans,Bartalinia laurinia, Bartalinia pondoensis, Bartalinia robillardoides,Beauveria bassiana, Bionectria ochroleuca, Bipolaris papendorfii,Boeremia exigua var. exigua, Botryosphaeria rhodina, Botrytis cinerea,Brachysporium nigrum, Cadophora (Phialophora) finlandica, Camarosporiumpalliatum, Camarosporium propinquum, Candida tropicalis, Capnodiumcoffeae, Ceratobasidium cornigerum, Ceratobasidium obscurum, Cercophoraterricola, Chaetomium globosum, Chaetomium sphaerale, Chaetosphaeriaendophytica, Chaetosphaeria ovoidea, Chaunopycnis alba, Chaunopycnispustulata, Chloridium phaeosporum, Chloridium preussii, Chromelosporiumfulvum, Cladorrhinum bulbillosum, Cladosporium cladosporioides,Cladosporium edgeworthrae, Cladosporium herbarum, Cladosporium orchidis,Cladosporium oxysporum, Cladosporium tenuissimum, Clonostachys rosea,Clonostachys rosea f. catenulate, Cochliobolus australiensis,Cochliobolus geniculatus, Cochliobolus hawaiiensis, Cochlioboluslunatus, Cochliobolus tuberculatus, Colletotrichum acutatum,Colletotrichum capsici, Colletotrichum crassipes, Colletotrichumdematium, Colletotrichum gloeosporioides, Colletotrichum magna,Colletotrichum musae, Colletotrichum orbiculare, Colletotrichumtruncatum, Coniella minima, Coniochaeta tetraspora, Coniochaetavelutina, Coniophora puteana, Coprinellus disseminates, Coprinellysradians, Cordyceps sinensis, Corynascus kuwaitiensis, Corynesporacassiicola, Crinipellis roreri, Cryphonectria parasitica, Cryptococcusvictoriae, Curvularia affinis, Curvularia oryzae, Curvulariasenegalensis, Curvularia sichuanensis, Cytosphaera mangiferae, Cytosporaeucalypticola, Daldinia eschscholzi., Davidiella tassiana, Debaryomyceshansenii, Deightoniella torulosa, Diaporthe cynaroidis, Diaporthe eres,Diaporthe helianthi, Diaporthe phaseolorum, Dictyochaeta triseptata,Dothiorella aromatics, Dothiorella dominicana, Drechslera ellisii,Elsinoe veneta, Embellisia eureka, Emericella nidulans, Engyodontiumalbum, Epicoccum nigrum, Epulorhiza anaticula, Epulorhiza repens,Eurotium amstelodami, Exserohilum rostratum, Fasciatispora petrakii,Fimetariella rabenhorstii, Fomes fomentarius, Fomes fomentarius,Fomitopsis ostreiformis, Fomitopsis pinicola, Fusarium anthophilum,Fusarium aquaeductuum, Fusarium avenaceum, Fusarium bulbicola, Fusariumchlamydosporum, Fusarium culmorum, Fusarium equiseti, Fusariumincarnatum, Fusarium lichenicola, Fusarium moniliforme, Fusariumoxysporum, Fusarium poae, Fusarium polyphialidicum, Fusariumproliferatum, Fusarium pulverosum, Fusarium semitectum var. majus,Fusarium solani, Fusarium sporotrichioides, Fusarium tricinctum,Fusarium verticillioides, Fusicladium britannicum, Ganoderma tsugae,Geomyces vinaceus, Gibberella avenacea, Gibberella baccata, Gibberellafujikuroi, Gibberella moniliformis, Gibberella zeae, Gliomastix murorum,Glomerella cingulata, Glomerella cingulate, Guignardi bidwelli,Guignardia camelliae, Guignardia citricarpa, Guignardia cocoicola,Guignardia mangiferae, Guignardia mangiferae, Guignardia vaccinii,Haematonectria haematococca, Haplotrichum minitissimum, Helgardiaanguioides, Helminthosporium chlorophorae, Hypocrea vixens, Hypoxylonfragiforme, Hypoxylon serpens, Hypoxylon stygium, Idriella amazonica,Idriella asaicola, Idriella euterpes, Idriella licualae, Ilyonectriaradicicola, Kabatiella caulivora, Kluyveromyces marxianus, Kretzschmariadeusta, Lasiodiplodia pseudotheobromae, Lasiodiplodia theobromae,Laspora coronate, Leiosphaerella cocoes, Lentinus squarrosulus,Lepteutypa cupressi, Leptosphaeria coniothyrium, Leptosphaerulinatrifolii, Letendraeopsis palmarum, Leucostoma niveum, Lewia eureka,Lewia eureka, Lunulospora curvula, Macrophomina phaseolina, Malbrancheacircinate, Massarina arundinariae, Melanospora zamiae, Melanotussubcuneiformis, Melanotus subcuneiformis, Microascus cinereus,Minimidochium setosum, Moniliopsis anomala, Monodictys levis, Morchellaelata, Mortierella alpine, Mucor fragilis, Mucor racemosus, Muscodoralbus, Mycena murina, Mycocentrospora acerina, Myriangium duriaei,Nectria haematococca, Nemania aenea, Nemania bipapillata, Nemaniaserpens, Neofusicoccum mangiferae, Neotyphodium lolii, Neurosporacrassa, Nigrospora oryzae, Nigrospora sphaerica, Nodulisporium anamorphof Hypoxylon fragiforme, Nodulisporium anamorph of Hypoxylon fuscum,Nodulisporium gregarium, Ochrocladosporium elatum, Ophiocordycepssobolifera, Ophiostoma stenoceras, Oxydothis poliothea, Paecilomycesformosus, Papulosa amerospora, Paraconiothyrium minitans,Paraphaeosphaeria quadriseptata, Penicillium biourgeianum, Penicilliumbrevicompactum, Peniophora cinerea, Periconia anamorph of Didymosphaeriaigniaria, Periconia digitata, Periconia hispidula, Periconia prolifica,Pestalotiopsis adusta, Pestalotiopsis caudata, Pestalotiopsis guepinii,Pestalotiopsis maculiformans, Pestalotiopsis microspora, Pestalotiopsispalmarum, Pestalotiopsis versicolor, Petriella sordida, Peziza varia,Peziza vesiculosa, Phaeangium lefebvrei, Phaedothis winteri,Phaeomoniella chlamydospora, Phaeotrichoconis crotalariae, Phanerochaeteaffinis, Phanerochaete sordida, Phialemonium dimorphosporum, Phlebiaradiate, Phlogicylindrium eucalypti, Phoma glomerate, Phoma herbarum,Phoma leveillei, Phoma moricola, Phoma radicina, Phoma sorghina, Phomasubglomerata, Phoma tracheiphila, Phoma tropica, Phomatosporabellaminuta, Phomatospora berkeleyi, Phomopsis anacardii, Phomopsiscasuarinae, Phomopsis leptostromiformis, Phomopsis mangiferae, Phomopsismanilkarae, Phomopsis orchidophila, Phyllosticta capitalensis,Phyllosticta colocasiicola, Phyllosticta minima, Phyllosticta sapotae,Piptarthron macrosporum, Piricauda pelagica, Piriformospora indica,Plagiostoma euphorbiae, Plenodomus fuscomaculans, Pleurophoma cava,Pleurotus ostreatus, Podospora fimbriata, Porosphaerella borinquensis,Preussia mediterranea, Preussia minima, Pseudocercospora punicae,Pseudocochliobolus pallescens, Pycnoporus cinnabarinus, Pycnoporussanguineus, Pyriculariopsis parasitica, Ramichloridium apiculatum,Ramichloridium biverticillatum, Rhizopus stolonifer, Rhizopycnis vagum,Rhizosphaera kalkhoffii, Rhodotorula minuta, Schizophyllum commune,Scolecobasidium terreum, Scolicotrichum musae, Scopuloides hydnoides,Scytalidium lignicola, Sebacina vermifera, Septoria anacardii,Setosphaeria rostrata, Sordaria fimicola, Sordaria tomento-alba,Sporormiella minima, Stagonosporopsis dorenboschii, Stemphyliumbotryosum, Stemphylium solani, Stilbohypoxylon quisquiliarum var.quisquiliarum, Streptomyces albosporus, Streptomyces aureus,Streptomyces cinereus, Streptomyces glaucus, Streptomyces globisporus,Streptomyces griseofuscus, Streptomyces griseorubroviolaceus,Streptomyces hygroscopicus, Streptomyces roseosporus, Sydowia polyspora,Talaromyces flavus, Talaromyces ohiensis, Talaromyces ohiensis,Tetracladium furcatum, Thanatephorus cucumeris, Thanatephorus pennatus,Thermomyces lanuginosus, Thumenella cubispora, Torula herbarum fquaternella, Trametes hirsuta, Trematosphaeria pertusa, Trichodermahamatum, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma viride, Trichothecium roseum,Triscelophorus acuminatus, Triscelophorus konajensis, Triscelophorusmonosporus, Truncatella angustata, Truncatella conorum-piceae,Tulasnella calospora, Ulocladium atrum, Ulocladium cucurbitae, Ustilagowilliamsii, Valsa ceratosperma, Verruculina enalia, Verticilliumlecanii, Wiesneriomyces laurinus, Wrightoporia tropicalis, Xylariaacuta, Xylaria adscendens, Xylaria allantoidea, Xylaria anisopleura,Xylaria arbuscula, Xylaria castorea Berk., Xylaria coccophora, Xylariacubensis, Xylaria curta, Xylaria hypoxylon, Xylaria microceras, Xylariamultiplex, Xylaria obovata, Xylaria palmicola, Xylaria telfairii,Zalerion maritimum, Zygosporium echinosporum, Zygosporium gibbum.

In one embodiment, the endophytic fungus is of a family selected fromthe group consisting of: Cladosporiaceae, Gnomoniaceae, Incertae sedis,Lasiosphaeriaceae, Netriaceae, Pleosporaceae.

In one embodiment, the endophytic fungus is of a genus selected from thegroup consisting of: Acremonium, Alternaria, Cladosporium, Cochliobolus,Embellisia, Epicoccum, Fusarium, Nigrospora, Phoma, and Podospora. Insome embodiments, the endophytic fungus is of species Acremoniumstrictum, Acremonium zeae, Alternaria alternate, Alternaria epidermidis,Alternaria infectoria, Alternaria tenuissima, Cladosporium tenuissimum,Epicoccum nigrum, Epicoccum sorghinum, Fusarium nigrum, Fusarium udum,Fusarium verticillioides, and Nigrospora oryza.

In one embodiment, the endophytic bacterium comprising in its genome anucleic acid sequence selected from the group consisting of: SEQ ID NOs:3597, 3602, 3605, 3610, 3611, 3612, 3613, 3614, 3615, 3616, 3617, 3618,3640, 3642, 3643, 3644, 3647, 3650, 3654, 3655, 3657, 3658, 3659, 3660,3661, 3662, 3672, 3673, 3674, 3675, 3676, 3677, 3678, 3679, 3680, 3681,3682, 3683, 3684, 3685, 3686, 3687, 3688, 3689, 3690, 3691, 3692, 3693,3694, 3695, 3696, 3697, 3698, 3699, 3700.

In one embodiment, the endophytic fungus comprises in its genome anucleic acid sequence that is at least 90% identical, at least 91%identical, at least 92% identical, at least 93% identical, at least 94%identical, at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, at least 99% identical, at least99.5% identical or 100% identical to a sequence selected from the groupconsisting of: SEQ ID NOs: 3597, 3602, 3605, 3610, 3611, 3612, 3613,3614, 3615, 3616, 3617, 3618, 3640, 3642, 3643, 3644, 3647, 3650, 3654,3655, 3657, 3658, 3659, 3660, 3661, 3662, 3672, 3673, 3674, 3675, 3676,3677, 3678, 3679, 3680, 3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688,3689, 3690, 3691, 3692, 3693, 3694, 3695, 3696, 3697, 3698, 3699, 3700.

In one embodiment, the endophyte comprises comprising in its genome anucleic acid sequence a nucleic acid sequence that is at least 90%identical, for example, at least 91% identical, at least 92% identical,at least 93% identical, at least 94% identical, at least 95% identical,at least 96% identical, at least 97% identical, at least 98% identical,at least 99% identical, at least 99.5% identical or 100% identical, to anucleic acid sequences found among the group consisting of SEQ ID NOs:1-3700.

In some aspects, the endophyte of any method or composition of thepresent invention comprises a nucleic acid sequence that is at least 90%identical, for example, at least 91% identical, at least 92% identical,at least 93% identical, at least 94% identical, at least 95% identical,at least 96% identical, at least 97% identical, at least 98% identical,at least 99% identical, at least 99.5% identical or 100% identical toany nucleic acid provided in Tables 1A, 2A, 3A, 4A, 5-14, 16-23.

Exogenous Endophytes.

In one embodiment, the endophyte is an endophytic microbe that wasisolated from a different plant than the inoculated plant. For example,in one embodiment, the endophyte can be an endophyte isolated from adifferent plant of the same species as the inoculated plant. In somecases, the endophyte can be isolated from a species related to theinoculated plant.

The breeding of plants for agriculture, their propagation in alteredenvironments, as well as cultural practices used to combat microbialpathogens, may have resulted in the loss in modern cultivars of theendophytes present in their wild ancestors, or such practices may haveinadvertently promoted other novel or rare plant-endophyte interactions,or otherwise altered the microbial population. The former is the case inmaize and its phylogenetically confirmed, direct wild ancestor,Parviglumis teosinte (Zea mays ssp. Parviglumis). It is possible thatthis higher diversity and titer of endophytes in the ancestor iscorrelated with an equally wide range of physiological responses derivedfrom the symbiosis that allow the plant to better adapt to theenvironment and tolerate stress. In order to survey plant groups forpotentially useful endophytes, seeds of their wild ancestors, wildrelatives, primitive landraces, modern landraces, modern breeding lines,and elite modern agronomic varieties can be screened for microbialendophytes by culture and culture independent methods as describedherein.

In some cases, plants are inoculated with endophytes that are exogenousto the seed of the inoculated plant. In one embodiment, the endophyte isderived from a plant of another species. For example, an endophyte thatis normally found in dicots is applied to a monocot plant (e.g.,inoculating corn with a soy bean-derived endophyte), or vice versa. Inother cases, the endophyte to be inoculated onto a plant can be derivedfrom a related species of the plant that is being inoculated. In oneembodiment, the endophyte can be derived from a related taxon, forexample, from a related species. The plant of another species can be anagricultural plant. For example, an endophyte derived from Hordeumirregulare can be used to inoculate a Hordeum vulgare L., plant.Alternatively, it can be derived from a ‘wild’ plant (i.e., anon-agricultural plant). For example, endophytes normally associatedwith the wild cotton Gossypium klotzschianum can be used to inoculatecommercial varieties of Gossypium hirsutum plants. As an alternativeexample of deriving an endophyte from a ‘wild’ plant, endophyticbacteria isolated from the South East Asian jungle orchid, Cymbidiumeburneum, can be isolated and testing for their capacity to benefitseedling development and survival of agricultural crops such as wheat,maize, soy and others [Facia, D. C., et al., (2013) World Journal ofMicrobiology and Biotechnology. 29(2). pp. 217-221]. In other cases, theendophyte can be isolated from an ancestral species of the inoculatedplant. For example, an endophyte derived from Zea diploperennis can beused to inoculate a commercial variety of modern corn, or Zea mays.

Relocalization of Endophytes.

While the endophyte that is coated onto the surface of the seed of thefirst plant is isolated from inside the seed of the second plant, theendophyte can relocalize to other tissues or plant parts once the seedof the first plant germinates. As such, in one embodiment, the seedendophyte which is coated onto the seed of a plant is capable, upongermination of the seed into a vegetative state, of localizing to adifferent tissue of the plant. For example, the endophyte can be capableof localizing to any one of the tissues in the plant, including: theroot, adventitious root, seminal root, root hair, shoot, leaf, flower,bud, tassel, meristem, pollen, pistil, ovaries, stamen, fruit, stolon,rhizome, nodule, tuber, trichome, guard cells, hydathode, petal, sepal,glume, rachis, vascular cambium, phloem, and xylem. In one embodiment,the endophyte is capable of localizing to the root and/or the root hairof the plant. In another embodiment, the endophyte is capable oflocalizing to the photosynthetic tissues, for example, leaves and shootsof the plant. In other cases, the endophyte is localized to the vasculartissues of the plant, for example, in the xylem and phloem. In stillanother embodiment, the endophyte is capable of localizing to thereproductive tissues (flower, pollen, pistil, ovaries, stamen, fruit) ofthe plant. In another embodiment, the endophyte is capable of localizingto the root, shoots, leaves and reproductive tissues of the plant. Instill another embodiment, the endophyte colonizes a fruit or seed tissueof the plant. In still another embodiment, the endophyte is able tocolonize the plant such that it is present in the surface of the plant(i.e., its presence is detectably present on the plant exterior, or theepisphere of the plant). In still other embodiments, the endophyte iscapable of localizing to substantially all, or all, tissues of theplant. In certain embodiments, the endophyte is not localized to theroot of a plant. In other cases, the endophyte is not localized to thephotosynthetic tissues of the plant.

Compositions Comprising Endophytes

In some embodiments, the endophyte is capable of metabolizing D-alanine,D-aspartic acid, D-serine, D-threonine, glycyl-L-aspartic acid,glycyl-L-glutamic acid, glycyl-L-proline, glyoxylic acid, inosine,L-alanine, L-alanyl-glycine, L-arabinose, L-asparagine, L-aspartic acid,L-glutamic acid, L-glutamine, L-proline, L-serine, L-threonine,tyramine, uridine, proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, and salicin. In oneembodiment, the endophyte comprises in its genome a nucleic acidsequence encoding a protein allowing it to metabolize arabinose. In oneembodiment, the protein is selected from the group consisting of SEQ IDNO: 3701-3813.

In some embodiments, the plant or plant element experiences an improvedability to grow in water-limited conditions, as a result of beingassociated with the endophyte or endophyte combinations.

In still another embodiment, the plant element of the first plant can befrom a genetically modified plant. In another embodiment, the plantelement of the first plant can be a hybrid plant element.

The synthetic combination can comprise a plant element of the firstplant which is surface-sterilized prior to combining with theendophytes.

As stated above, the endophyte used in the synthetic combination isderived from a plant element of a second plant. In one embodiment, thesecond plant is a monocotyledonous plant or tissue thereof. In aparticular embodiment, the second plant is a cereal plant or tissuethereof. In yet another embodiment, the second plant is selected fromthe group consisting of a maize plant, a barley plant, a wheat plant, asugarcane plant, or a rice plant. In one embodiment, the plant elementis a naked grain (i.e., without hulls or fruit cases). In an alternativeembodiment, the second plant is a dicotyledonous plant. For example, thesecond plant can be selected from the group consisting of a cottonplant, a tomato plant, a pepper plant, or a soybean plant.

The synthetic combination can additionally comprise a seed coatingcomposition, a root treatment, or a foliar application composition. Theseed coating composition, or the root treatment, or the foliarapplication composition can comprise an agent selected from the groupconsisting of: a fungicide, an antibacterial agent, an herbicide, anematicide, an insecticide, a plant growth regulator, a rodenticide anda nutrient, or a combination thereof. The seed coating composition, orthe root treatment, or the foliar application composition can furthercomprise an agent selected from the group consisting of anagriculturally acceptable carrier, a tackifier, a microbial stabilizer,or a combination thereof. In still another embodiment, the seed coatingcomposition, or the root treatment, or the foliar applicationcomposition can contain a second microbial preparation, including butnot limited to a rhizobial bacterial preparation.

The present invention contemplates the use of endophytes that areunmodified, as well as those that are modified. In one embodiment, theendophyte is a recombinant endophyte. In one particular embodiment, theendophyte is modified prior to coating onto the surface of the plantelement such that it has enhanced compatibility with an antimicrobialagent when compared with the unmodified. For example, the endophyte canbe modified such that it has enhanced compatibility with anantibacterial agent. In an alternative embodiment, the endophyte hasenhanced compatibility with an antifungal agent. The endophyte can bemodified such that it exhibits at least 3 fold greater, for example, atleast 5 fold greater, at least 10 fold greater, at least 20 foldgreater, at least 30 fold greater or more resistance to an antimicrobialagent when compared with the unmodified endophyte.

The present invention also contemplates the use of multiple endophytes.For example, in one embodiment, the synthetic combination describedabove can comprise a plurality of purified endophytes, for example, 2,3, 4 or more different types of endophytes.

In one embodiment, the formulation comprises an endophyte that comprisesa nucleic acid sequence that is at least 97% identical, for example, atleast 98% identical, at least 99% identical, at least 99.5% identical,or 100% identical, to any nucleic acid selected from SEQ ID NOs: 1-3700.

In one embodiment, the formulation comprises at least two endophyticmicrobial entities that separately comprise a nucleic acid sequence thatis at least 97% identical, for example, at least 98% identical, at least99% identical, at least 99.5% identical, or 100% identical, to anynucleic acid selected from SEQ ID NOs: 1-3700.

In one embodiment, the agronomic trait is selected from the groupconsisting of altered oil content, altered protein content, altered seedcarbohydrate composition, altered seed oil composition, and altered seedprotein composition, chemical tolerance, cold tolerance, delayedsenescence, disease resistance, drought tolerance, ear weight, growthimprovement, health enhancement, heat tolerance, herbicide tolerance,herbivore resistance, improved nitrogen fixation, improved nitrogenutilization, improved root architecture, improved water use efficiency,increased biomass, increased root length, increased seed weight,increased shoot length, increased yield, increased yield underwater-limited conditions, kernel mass, kernel moisture content, metaltolerance, number of ears, number of kernels per ear, number of pods,nutrition enhancement, pathogen resistance, pest resistance,photosynthetic capability improvement, salinity tolerance, stay-green,vigor improvement, increased dry weight of mature seeds, increased freshweight of mature seeds, increased number of mature seeds per plant,increased chlorophyll content, increased number of pods per plant,increased length of pods per plant, reduced number of wilted leaves perplant, reduced number of severely wilted leaves per plant, and increasednumber of non-wilted leaves per plant, a detectable modulation in thelevel of a metabolite, a detectable modulation in the level of atranscript, and a detectable modulation in the proteome relative to areference plant. In another embodiment, at least two agronomic traitsare improved in the agricultural plant.

In another embodiment, the formulation comprising the endophyte isdisposed in an amount effective to detectably increase the rate ofgermination of the seed. For example, the rate of germination of theseed is increased by at least 0.5%, for example, at least 1%, at least2%, at least 3%, at least 5%, at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100% or more, when compared with a referenceagricultural plant.

In another embodiment, the formulation comprising the endophyte isdisposed in an amount effective to detectably increase the biomass ofthe plant. For example, the biomass of the plant is detectably increasedby at least 1%, for example, at least 2%, at least 3%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 100%, or more, when compared with a reference agricultural plant.

In another embodiment, the formulation comprising the endophyte isdisposed in an amount effective to increase the biomass or yield of afruit or seed of the plant. For example, the biomass of the fruit orseed of the plant is detectably increased by at least 1%, for example,at least 2%, at least 3%, at least 5%, at least 10%, at least 15%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, or more, whencompared with the fruit or seed of a reference agricultural plant.

In still another embodiment, the formulation comprising the endophyte isdisposed in an amount effective to increase the height of the plant. Theheight of the plant, in some embodiments, is detectably increased by atleast 1%, for example, at least 2%, at least 3%, at least 5%, at least10%, at least 15%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least100%, or more, when compared with the height of a reference agriculturalplant.

Combinations of Endophytes

In another embodiment, the present invention contemplates methods ofassociating a plurality of endophytes with one or more plant elements,such as a seed, a leaf, or a root, in order to confer an improvedagronomic trait or improved agronomic trait potential to said plantelement or host plant.

In some embodiments, the invention contemplates coating the seed of aplant with a plurality of endophytes, as well as seed compositionscomprising a plurality of endophytes on and/or in the seed. The methodsaccording to this embodiment can be performed in a manner similar tothose described herein for single endophyte coating. In one example,multiple endophytes can be prepared in a single preparation that iscoated onto the seed. The endophytes can be from a common origin (i.e.,a same plant). Alternatively, the endophytes can be from differentplants. Thus, the present invention provides for combinations comprisingat least two endophytic microbial populations with an agriculturalplant. The endophytic populations are heterologously disposed on anexterior surface of or within the plant in an amount effective tocolonize the plant. The combination can further comprise a formulationthat comprises at least one member selected from the group consisting ofan agriculturally compatible carrier, a tackifier, a microbialstabilizer, a fungicide, an antibacterial agent, an herbicide, anematicide, an insecticide, a plant growth regulator, a rodenticide, anda nutrient.

Where multiple endophytes are coated onto the seed of the plant, eachendophyte can be a bacterium. In the alternative, each endophyte can bea fungus. In still another embodiment, a mixture of bacterial and fungalendophytes can be coated onto the surface of a seed.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, orat least ten or more endophytes of an endophytic combination is abacterium selected from one of the following genera: Acidovorax,Agrobacterium, Bacillus, Burkholderia, Chryseobacterium, Curtobacterium,Enterobacter, Escherichia, Methylobacterium, Paenibacillus, Pantoea,Pseudomonas, Ralstonia, Saccharibacillus, Sphingomonas, andStenotrophomonas.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, orat least ten or more endophytes of an endophytic combination is a fungusselected from one of the following genera: Acremonium, Alternaria,Cladosporium, Cochliobolus, Embellisia, Epicoccum, Fusarium, Nigrospora,Phoma, and Podospora.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, orat least ten or more endophytes of an endophytic combination is abacterium selected from one of the following families: Bacillaceae,Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae,Methylobacteriaceae, Microbacteriaceae, Paenibacillileae,Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, orat least ten or more endophytes of an endophytic combination is a fungusselected from one of the following families: Cladosporiaceae,Gnomoniaceae, Incertae sedis, Lasiosphaeriaceae, Netriaceae,Pleosporaceae.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, orat least ten or more endophytes of an endophytic combination areselected from one of the following genera: Acidovorax, Agrobacterium,Bacillus, Burkholderia, Chryseobacterium, Curtobacterium, Enterobacter,Escherichia, Methylobacterium, Paenibacillus, Pantoea, Pseudomonas,Ralstonia, Saccharibacillus, Sphingomonas, and Stenotrophomonas,Acremonium, Alternaria, Cladosporium, Cochliobolus, Embellisia,Epicoccum, Fusarium, Nigrospora, Phoma, and Podospora.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, orat least ten or more endophytes of an endophytic combination areselected from one of the following families: Bacillaceae,Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae,Methylobacteriaceae, Microbacteriaceae, Paenibacillileae,Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae,Cladosporiaceae, Gnomoniaceae, Incertae sedis, Lasiosphaeriaceae,Netriaceae, Pleosporaceae.

In one embodiment, at least one of the endophytic populations comprise anucleic acid that is at least 90% identical, at least 91% identical, atleast 92% identical, at least 93% identical, at least 94% identical, atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, at least 99% identical, at least 99.5% identical,or 100% identical to a sequence selected from the group consisting of:SEQ ID NOs: 1-3700.

In some embodiments, the combination of endophytes comprises at leasttwo, at least three, at least four, at least five, or greater than five,endophytes, each comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NO: 1-3700.

In some embodiments, the combination of endophytes comprises at leasttwo, at least three, at least four, at least five, or greater than five,endophytes, each comprising a nucleic acid sequence at least 80%, atleast 85%, at least 90%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical from a sequence selected from the group consisting of SEQID NO: 1-3700.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, orat least ten or more endophytes of an endophytic combination is abacterium comprising in its genome a nucleic acid sequence selected fromSEQ ID NOs: 3588, 3589, 3590, 3591, 3592, 3593, 3594, 3595, 3596, 3598,3599, 3600, 3601, 3603, 3604, 3606, 3607, 3608, 3609, 3619, 3620, 3621,3622, 3623, 3624, 3625, 3626, 3627, 3628, 3629, 3630, 3631, 3632, 3633,3634, 3635, 3636, 3637, 3638, 3639, 3641, 3645, 3646, 3648, 3649, 3651,3652, 3653, 3656, 3663, 3664, 3665, 3666, 3667, 3668, 3669, 3670, 3671.In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, orat least ten or more endophytes of an endophytic combination is a funguscomprising in its genome a nucleic acid sequence selected from SEQ IDNOs: 3597, 3602, 3605, 3610, 3611, 3612, 3613, 3614, 3615, 3616, 3617,3618, 3640, 3642, 3643, 3644, 3647, 3650, 3654, 3655, 3657, 3658, 3659,3660, 3661, 3662, 3672, 3673, 3674, 3675, 3676, 3677, 3678, 3679, 3680,3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688, 3689, 3690, 3691, 3692,3693, 3694, 3695, 3696, 3697, 3698, 3699, 3700. In one embodiment, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least night, or at least ten or moreendophytes of an endophytic combination are endophytes each comprisingin its genome a nucleic acid sequence selected from the group consistingof: SEQ ID NOs: 3588, 3589, 3590, 3591, 3592, 3593, 3594, 3595, 3596,3598, 3599, 3600, 3601, 3603, 3604, 3606, 3607, 3608, 3609, 3619, 3620,3621, 3622, 3623, 3624, 3625, 3626, 3627, 3628, 3629, 3630, 3631, 3632,3633, 3634, 3635, 3636, 3637, 3638, 3639, 3641, 3645, 3646, 3648, 3649,3651, 3652, 3653, 3656, 3663, 3664, 3665, 3666, 3667, 3668, 3669, 3670,3671, 3597, 3602, 3605, 3610, 3611, 3612, 3613, 3614, 3615, 3616, 3617,3618, 3640, 3642, 3643, 3644, 3647, 3650, 3654, 3655, 3657, 3658, 3659,3660, 3661, 3662, 3672, 3673, 3674, 3675, 3676, 3677, 3678, 3679, 3680,3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688, 3689, 3690, 3691, 3692,3693, 3694, 3695, 3696, 3697, 3698, 3699, 3700.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, orat least ten or more endophytes of an endophytic combination is abacterium comprising in its genome a nucleic acid that is at least 90%identical, at least 91% identical, at least 92% identical, at least 93%identical, at least 94% identical, at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, at least 99%identical, at least 99.5% identical, or 100% identical to a sequenceselected from the group consisting of: SEQ ID NOs: 3588, 3589, 3590,3591, 3592, 3593, 3594, 3595, 3596, 3598, 3599, 3600, 3601, 3603, 3604,3606, 3607, 3608, 3609, 3619, 3620, 3621, 3622, 3623, 3624, 3625, 3626,3627, 3628, 3629, 3630, 3631, 3632, 3633, 3634, 3635, 3636, 3637, 3638,3639, 3641, 3645, 3646, 3648, 3649, 3651, 3652, 3653, 3656, 3663, 3664,3665, 3666, 3667, 3668, 3669, 3670, 3671. In one embodiment, at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least night, or at least ten or moreendophytes of an endophytic combination is a fungus comprising in itsgenome a nucleic acid that is at least 90% identical, at least 91%identical, at least 92% identical, at least 93% identical, at least 94%identical, at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, at least 99% identical, at least99.5% identical, or 100% identical to a sequence selected from the groupconsisting of: SEQ ID NOs: 3597, 3602, 3605, 3610, 3611, 3612, 3613,3614, 3615, 3616, 3617, 3618, 3640, 3642, 3643, 3644, 3647, 3650, 3654,3655, 3657, 3658, 3659, 3660, 3661, 3662, 3672, 3673, 3674, 3675, 3676,3677, 3678, 3679, 3680, 3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688,3689, 3690, 3691, 3692, 3693, 3694, 3695, 3696, 3697, 3698, 3699, 3700.In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least night, orat least ten or more endophytes of an endophytic combination areendophytes each comprising in its genome a nucleic acid that is at least90% identical, at least 91% identical, at least 92% identical, at least93% identical, at least 94% identical, at least 95% identical, at least96% identical, at least 97% identical, at least 98% identical, at least99% identical, at least 99.5% identical, or 100% identical to a sequenceselected from the group consisting of: SEQ ID NOs: 3588, 3589, 3590,3591, 3592, 3593, 3594, 3595, 3596, 3598, 3599, 3600, 3601, 3603, 3604,3606, 3607, 3608, 3609, 3619, 3620, 3621, 3622, 3623, 3624, 3625, 3626,3627, 3628, 3629, 3630, 3631, 3632, 3633, 3634, 3635, 3636, 3637, 3638,3639, 3641, 3645, 3646, 3648, 3649, 3651, 3652, 3653, 3656, 3663, 3664,3665, 3666, 3667, 3668, 3669, 3670, 3671, 3597, 3602, 3605, 3610, 3611,3612, 3613, 3614, 3615, 3616, 3617, 3618, 3640, 3642, 3643, 3644, 3647,3650, 3654, 3655, 3657, 3658, 3659, 3660, 3661, 3662, 3672, 3673, 3674,3675, 3676, 3677, 3678, 3679, 3680, 3681, 3682, 3683, 3684, 3685, 3686,3687, 3688, 3689, 3690, 3691, 3692, 3693, 3694, 3695, 3696, 3697, 3698,3699, 3700.

In some aspects, the combination of endophytes comprises at least twoendophytes that each comprise at least one nucleic acid sequence that isat least 90% identical, for example, at least 91% identical, at least92% identical, at least 93% identical, at least 94% identical, at least95% identical, at least 96% identical, at least 97% identical, at least98% identical, at least 99% identical, at least 99.5% identical or 100%identical to any nucleic acid provided in Tables 1A, 2A, 3A, 4A, 5-14,16-23.

In some aspects, the combination of endophytes comprises at least twoendophytes provided in any of Tables 2B, 3B, 4B, and 15.

Where multiple endophytes are coated onto the seed, any or all of theendophytes may be capable of conferring a beneficial trait onto the hostplant. In some cases, all of the endophytes are capable of conferring abeneficial trait onto the host plant. The trait conferred by each of theendophytes may be the same (e.g., both improve the host plant'stolerance to a particular biotic stress), or may be distinct (e.g., oneimproves the host plant's tolerance to drought, while another improvesphosphate utilization). In other cases the conferred trait may be theresult of interactions between the endophytes.

Combinations of endophytes can be selected by any one or more of severalcriteria. In one embodiment, compatible endophytes are selected. As usedherein, “compatibility” refers to endophyte populations that do notsignificantly interfere with the growth, propagation, and/or productionof beneficial substances of the other. Incompatible endophytepopulations can arise, for example, where one of the populationsproduces or secrets a compound that is toxic or deleterious to thegrowth of the other population(s). Incompatibility arising fromproduction of deleterious compounds/agents can be detected using methodsknown in the art, and as described herein elsewhere. Similarly, thedistinct populations can compete for limited resources in a way thatmakes co-existence difficult.

In another embodiment, combinations are selected on the basis ofcompounds produced by each population of endophytes. For example, thefirst population is capable of producing siderophores, and anotherpopulation is capable of producing anti-fungal compounds. In oneembodiment, the first population of endophytes or endophytic componentsis capable of a function selected from the group consisting of auxinproduction, nitrogen fixation, production of an antimicrobial compound,siderophore production, mineral phosphate solubilization, cellulaseproduction, chitinase production, xylanase production, and acetoinproduction. In another embodiment, the second population of endophytesor endophytic component is capable of a function selected from the groupconsisting of auxin production, nitrogen fixation, production of anantimicrobial compound, siderophore production, mineral phosphatesolubilization, cellulase production, chitinase production, xylanaseproduction, and acetoin production. In still another embodiment, thefirst and second populations are capable of at least one differentfunction.

In another embodiment, combinations are selected on the basis of carbonsources they metabolize. In some aspects, an endophyte may be capable ofusing any one or more of the following: 1,2-Propanediol, 2-Aminoethanol,2-Deoxy adenosine, Acetic acid, Acetoacetic acid, Adenosine, Adonitol,Bromo succinic acid, Citric acid, D-Alanine, D-Aspartic acid,D-Cellobiose, D-Fructose, D-Fructose-6-Phosphate, D-Galactonicacid-γ-lactone, D-Galactose, D-Galacturonic acid, D-Gluconic acid,D-Glucosaminic acid, D-Glucose-1-Phosphate, D-Glucose-6-Phosphate,D-Glucuronic acid, D-L-Malic acid, D-L-α-Glycerol phosphate, D-Malicacid, D-Mannitol, D-Mannose, D-Melibiose, D-Psicose, D-Ribose,D-Saccharic acid, D-Serine, D-Sorbitol, D-Threonine, D-Trehalose,Dulcitol, D-Xylose, Formic acid, Fumaric acid, Glucuronamide, Glycerol,Glycolic acid, Glycyl-L-Aspartic acid, Glycyl-L-Glutamic acid,Glycyl-L-Proline, Glyoxylic acid, Inosine, Lactulose, L-Alanine,L-Alanyl-Glycine, L-Arabinose, L-Asparagine, L-Aspartic acid, L-Fucose,L-Galactonic-acid-α-lactone, L-Glutamic acid, L-glutamine, L-Lacticacid, L-Lyxose, L-Malic acid, L-Proline, L-Rhamnose, L-Serine,L-Threonine, Maltose, Maltotriose, Methyl Pyruvate, m-Hydroxy PhenylAcetic acid, m-Inositol, Mono Methyl Succinate, m-Tartaric acid, Mucicacid, N-acetyl-β-D-Mannosamine, N-Acetyl-D-Glucosamine,Phenylethyl-amine, p-Hydroxy Phenyl acetic acid, Propionic acid, Pyruvicacid, Succinic acid, Sucrose, Thymidine, Tricarballylic acid, Tween 20,Tween 40, Tween 80, Tyramine, Uridine, α-D-Glucose, α-D-Lactose,α-Hydroxy Butyric acid, α-Hydroxy Glutaric acid-γ-lactone,α-Keto-Butyric acid, α-Keto-Glutaric acid, α-Methyl-D-Galactoside,β-Methyl-D-glucoside. In preferred embodiments, at least one populationis capable of metabolizing any one or more of the following: D-Alanine,D-Aspartic acid, D-Serine, D-ThreonineGlycyl-L-Aspartic acid,Glycyl-L-Glutamic acid, Glycyl-L-Proline, Glyoxylic acid, Inosine,L-Alanine, L-Alanyl-Glycine, L-Arabinose, L-Asparagine, L-Aspartic acid,L-Glutamic acid, L-glutamine, L-Proline, L-Serine, L-Threonine,Tyramine, Uridine, Proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, salicin.

In another aspect, the combination of endophytes comprises at least oneendophyte that is capable of metabolizing any one or more of thefollowing: D-Alanine, D-Aspartic acid, D-Serine,D-ThreonineGlycyl-L-Aspartic acid, Glycyl-L-Glutamic acid,Glycyl-L-Proline, Glyoxylic acid, Inosine, L-Alanine, L-Alanyl-Glycine,L-Arabinose, L-Asparagine, L-Aspartic acid, L-Glutamic acid,L-glutamine, L-Proline, L-Serine, L-Threonine, Tyramine, Uridine,Proline, arabinose, xylose, mannose, sucrose, maltose, D-glucosamine,trehalose, oxalic acid, salicin.

For example, one endophyte may be capable of utilizing oxalic acid and asecond endophyte may be capable of using arabinose. For example, atleast one endophyte may be capable of metabolizing proline, at least oneendophyte may be capable of metabolizing mannose. It is contemplatedthat combinations of endophytes may be selected based on complementarymetabolic capabilities: for example, one may be capable of utilizingmannose but not sucrose, and a second may be capable of utilizingsucrose but not mannose. In another aspect, it is contemplated thatcombinations of endophytes may be selected based on mutual metaboliccapabilities: for example, two endophytes that both are able to utilizemannose. In another aspect, it is contemplated that combinations ofendophytes are selected based on the synergistic effects of carbonsource utilization: for example, one endophyte may have the capabilityof utilizing mannose but not proline but when in combination with asecond endophyte may then display the ability to utilize proline. Inother words, one endophyte may be able to promote the ability of anotherendophyte to utilize a particular carbon source. In another aspect, oneendophyte may reduce the ability of another endophyte to utilize aparticular carbon source. In another aspect of synergism, two endophytesthat are themselves each capable of utilizing one type of carbon source,for example, maltose, may enhance each others' abilities to utilize saidcarbon source at a greater efficiacy. It is contemplated that anycombination (mutual, complementary, additive, synergistic) of substrateutilization capabilities may be used as criteria of selection ofendophytes of the present invention. It is further contemplated thatsuch combinations of carbon substrate sources for endophyte utilizationmay include at least two, at least three, at least four, at least five,at least six, at least seven, at least eight, at least nine, at leastten, and even greater than ten different carbon sources.

In some embodiments, the combination of endophytes comprises at leasttwo, at least three, at least four, at least five, or greater than five,endophytes wherein at least one of said endophytes comprises a gene inits genome that encodes a protein selected from the group consisting of:arabinose ABC transporter ATP-binding protein, arabinose ABC transporterpermease, arabinose ABC transporter substrate-binding protein, arabinoseimport ATP-binding protein AraG, arabinose isomerase, arabinose-protonsymporter, L-arabinose ABC transporter periplasmic L-arabinose-bindingprotein, L-arabinose isomerase, L-arabinose transport ATP-bindingprotein araG, L-arabinose transporter ATP-binding protein, L-arabinosetransporter ATP-binding protein (plasmid), L-arabinose transporterpermease, L-arabinose transporter permease (plasmid), L-arabinosetransporter permease protein, L-arabinose-binding protein,arabinose-proton symporter.

In some embodiments, the combination of endophytes comprises at leasttwo, at least three, at least four, at least five, or greater than five,endophytes wherein each endophyte comprises a gene in its genome thatencodes a protein selected from SEQ ID NO: 3701-3913.

In some embodiments, the first endophyte comprises in its genome a genethat encodes a protein with at least 80%, at least 85%, at least 90%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical from a sequenceselected from the group consisting of SEQ ID NOs: 3701-3913

In some embodiments, the first endophyte comprises in its genome a genethat encodes a protein with at least 80%, at least 85%, at least 90%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical from a sequenceselected from the group consisting of SEQ ID NOs: 3701-3913, and thesecond endophyte comprises in its genome a gene that encodes a proteinwith at least 80% identity to a sequence selected from the groupconsisting of SEQ ID NOs: 3701-3913.

In some embodiments, the first population comprises in its genome a genethat encodes a protein with at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to a sequence selected from the group consisting of SEQ ID NOs:3701-3913. In some embodiments, the second population comprises in itsgenome a gene that encodes a protein with at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity to a sequence selected from the group consisting ofSEQ ID NOs: 3701-3913.

In some embodiments, the combination of endophytes comprises at leasttwo, at least three, at least four, at least five, or greater than five,endophytes capable of metabolizing at least one of the following:D-Alanine, D-Aspartic acid, D-Serine, D-ThreonineGlycyl-L-Aspartic acid,Glycyl-L-Glutamic acid, Glycyl-L-Proline, Glyoxylic acid, Inosine,L-Alanine, L-Alanyl-Glycine, L-Arabinose, L-Asparagine, L-Aspartic acid,L-Glutamic acid, L-glutamine, L-Proline, L-Serine, L-Threonine,Tyramine, Uridine, Proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, salicin.

In some embodiments, the combination of endophytes comprises at leastone endophyte comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1-3700, and at least one endophyte that iscapable of metabolizing at least one of D-Alanine, D-Aspartic acid,D-Serine, D-ThreonineGlycyl-L-Aspartic acid, Glycyl-L-Glutamic acid,Glycyl-L-Proline, Glyoxylic acid, Inosine, L-Alanine, L-Alanyl-Glycine,L-Arabinose, L-Asparagine, L-Aspartic acid, L-Glutamic acid,L-glutamine, L-Proline, L-Serine, L-Threonine, Tyramine, Uridine,Proline, arabinose, xylose, mannose, sucrose, maltose, D-glucosamine,trehalose, oxalic acid, salicin.

In another embodiment, the combination of endophytes comprises at leastone endophyte comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1-3700, and at least one endophyte thatcomprises a gene in its genome a gene that encodes a protein selectedfrom SEQ ID NO: 3701-3913.

In another embodiment, the combination of endophytes comprises at leastone endophyte that is capable of metabolizing at least one of D-Alanine,D-Aspartic acid, D-Serine, D-ThreonineGlycyl-L-Aspartic acid,Glycyl-L-Glutamic acid, Glycyl-L-Proline, Glyoxylic acid, Inosine,L-Alanine, L-Alanyl-Glycine, L-Arabinose, L-Asparagine, L-Aspartic acid,L-Glutamic acid, L-glutamine, L-Proline, L-Serine, L-Threonine,Tyramine, Uridine, Proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, salicin, and at leastone endophyte that comprises a gene in its genome a gene that encodes aprotein selected from SEQ ID NO: 3701-3913.

It is contemplated that each endophyte in the combination of endophytesmay comprise different characteristics, for example comprise genes withdifferent percent identities to any of the sequences of SEQS ID Nos:1-3913.

In still another embodiment, the combinations of endophytes are selectedfor their distinct localization in the plant after colonization. Forexample, the first population of endophytes or endophytic components cancolonize, and in some cases preferentially colonize, the root tissue,while a second population can be selected on the basis of itspreferential colonization of the aerial parts of the agricultural plant.Therefore, in one embodiment, the first population is capable ofcolonizing one or more of the tissues selected from the group consistingof a root, shoot, leaf, flower, and seed. In another embodiment, thesecond population is capable of colonizing one or more tissues selectedfrom the group consisting of root, shoot, leaf, flower, and seed. Instill another embodiment, the first and second populations are capableof colonizing a different tissue within the agricultural plant.

In still another embodiment, combinations of endophytes are selected fortheir ability to confer one or more distinct agronomic traits on theinoculated agricultural plant, either individually or in synergisticassociation with other endophytes. Alternatively, two or more endophytesinduce the colonization of a third endophyte. For example, the firstpopulation of endophytes or endophytic components is selected on thebasis that it confers significant increase in biomass, while the secondpopulation promotes increased drought tolerance on the inoculatedagricultural plant. Therefore, in one embodiment, the first populationis capable of conferring at least one trait selected from the groupconsisting of thermal tolerance, herbicide tolerance, droughtresistance, insect resistance, fungus resistance, virus resistance,bacteria resistance, male sterility, cold tolerance, salt tolerance,increased yield, enhanced nutrient use efficiency, increased nitrogenuse efficiency, increased tolerance to nitrogen stress, increasedfermentable carbohydrate content, reduced lignin content, increasedantioxidant content, enhanced water use efficiency, increased vigor,increased germination efficiency, earlier or increased flowering,increased biomass, altered root-to-shoot biomass ratio, enhanced soilwater retention, or a combination thereof. In another embodiment, thesecond population is capable of conferring a trait selected from thegroup consisting of thermal tolerance, herbicide tolerance, droughtresistance, insect resistance, fungus resistance, virus resistance,bacteria resistance, male sterility, cold tolerance, salt tolerance,increased yield, enhanced nutrient use efficiency, increased nitrogenuse efficiency, increased fermentable carbohydrate content, reducedlignin content, increased antioxidant content, enhanced water useefficiency, increased vigor, increased germination efficiency, earlieror increased flowering, increased biomass, altered root-to-shoot biomassratio, and enhanced soil water retention. In still another embodiment,each of the first and second population is capable of conferring adifferent trait selected from the group consisting of thermal tolerance,herbicide tolerance, drought resistance, insect resistance, fungusresistance, virus resistance, bacteria resistance, male sterility, coldtolerance, salt tolerance, increased yield, enhanced nutrient useefficiency, increased nitrogen use efficiency, increased fermentablecarbohydrate content, reduced lignin content, increased antioxidantcontent, enhanced water use efficiency, increased vigor, increasedgermination efficiency, earlier or increased flowering, increasedbiomass, altered root-to-shoot biomass ratio, and enhanced soil waterretention. In any combination of endophytes, any of the following traitsof agronomic importance may be modulated due to the association of oneor more of the endophytes in the combination with a plant or plantelement: altered oil content, altered protein content, altered seedcarbohydrate composition, altered seed oil composition, and altered seedprotein composition, chemical tolerance, cold tolerance, delayedsenescence, disease resistance, drought tolerance, ear weight, growthimprovement, health enhancement, heat tolerance, herbicide tolerance,herbivore resistance, improved nitrogen fixation, improved nitrogenutilization, improved root architecture, improved water use efficiency,increased biomass, increased root length, increased seed weight,increased shoot length, increased yield, increased yield underwater-limited conditions, kernel mass, kernel moisture content, metaltolerance, number of ears, number of kernels per ear, number of pods,nutrition enhancement, pathogen resistance, pest resistance,photosynthetic capability improvement, salinity tolerance, stay-green,vigor improvement, increased dry weight of mature seeds, increased freshweight of mature seeds, increased number of mature seeds per plant,increased chlorophyll content, increased number of pods per plant,increased length of pods per plant, reduced number of wilted leaves perplant, reduced number of severely wilted leaves per plant, and increasednumber of non-wilted leaves per plant, a detectable modulation in thelevel of a metabolite, a detectable modulation in the level of atranscript, or a detectable modulation in the proteome relative to areference plant.

The combinations of endophytes can also be selected based oncombinations of the above criteria. For example, the first population ofendophytes can be selected on the basis of the compound it produces(e.g., its ability to fix nitrogen, thus providing a potential nitrogensource to the plant), while the second population can be selected on thebasis of its ability to confer increased resistance of the plant to apathogen (e.g., a fungal pathogen).

In some aspects of the present invention, it is contemplated thatcombinations of endophytes can provide an increased benefit to the hostplant, as compared to that conferred by a single endophyte, by virtue ofadditive effects. For example, one endophyte strain that induces abenefit in the host plant may induce such benefit equally well in aplant that is also colonized with a different endophyte strain that alsoinduces the same benefit in the host plant. The host plant thus exhibitsthe same total benefit from the plurality of different endophyte strainsas the additive benefit to individual plants colonized with eachindividual endophyte of the plurality. In one example, a plant iscolonized with two different endophyte strains: one provides a 1×increase in biomass when associated with the plant, and the otherprovides a 2× increase in biomass when associated with a differentplant. When both endophyte strains are associated with the same plant,that plant would experience a 3× (additive of 1×+2× single effects)increase in auxin biomass. Additive effects are a surprising aspect ofthe present invention, as non-compatibility of endophytes may result ina cancellation of the beneficial effects of both endophytes.

In some aspects of the present invention, it is contemplated that acombination of endophytes can provide an increased benefit to the hostplant, as compared to that conferred by a single endophyte, by virtue ofsynergistic effects. For example, one endophyte strain that induces abenefit in the host plant may induce such benefit beyond additiveeffects in a plant that is also colonized with a different endophytestrain that also induces that benefit in the host plant. The host plantthus exhibits the greater total benefit from the plurality of differentendophyte strains than would be expected from the additive benefit ofindividual plants colonized with each individual endophyte of theplurality. In one example, a plant is colonized with two differentendophyte strains: one provides a 1× increase in biomass when associatedwith a plant, and the other provides a 2× increase in biomass whenassociated with a different plant. When both endophyte strains areassociated with the same plant, that plant would experience a 5×(greater than an additive of 1×+2× single effects) increase in biomass.Synergistic effects are a surprising aspect of the present invention.

Selection of Endophytes Conferring Beneficial Traits.

The present invention contemplates inoculation of plants with microbes.As described earlier, the microbes can be derived from many differentplants species, from different parts of the plants, and from plantsisolated across different environments. Once a microbe is isolated, itcan be tested for its ability to confer a beneficial trait. Numeroustests can be performed both in vitro and in vivo to assess whatbenefits, if any, are conferred upon the plant. In one embodiment, amicrobe is tested in vitro for an activity selected from the groupconsisting of: liberation of complexed phosphates, liberation ofcomplexed iron (e.g., through secretion of siderophores), production ofphytohormones, production of antibacterial compounds, production ofantifungal compounds, production of insecticidal compounds, productionof nematicidal compounds, production and/or secretion of ACC deaminase,production and/or secretion of acetoin, production and/or secretion ofpectinase, production and/or secretion of cellulase, and productionand/or secretion of RNAse. Exemplary in vitro methods for the above canbe found in the Examples sections below.

It is noted that the initial test for the activities listed above canalso be performed using a mixture of microbes, for example, a communityof microbes isolated from a single plant. A positive activity readoutusing such mixture can be followed with the isolation of individualmicrobes within that population and repeating the in vitro tests for theactivities to isolate the microbe responsible for the particularactivity. Once validated using a single microbe isolate, then the plantcan be inoculated with a microbe, and the test performed in vivo, eitherin growth chamber or greenhouse conditions, and comparing with a controlplant that was not inoculated with the microbe.

It is contemplated that each endophyte in the combination of endophytesmay comprise different characteristics, for example but not limited to:comprise genes with different percent identities to any of the sequencesof SEQS ID Nos: 1-3913, different phenotypic characteristics, differentabilities to utilize various carbon sources, different abilities toconfer agronomic trait potentials or improved agronomic traits to a hostseed or plant to which it may become associated, different localizationin plant elements.

Plants Useful for the Present Invention

The methods and compositions according to the present invention can bedeployed for any seed plant species. Thus, the invention has use over abroad range of plants, preferably higher plants pertaining to theclasses of Angiospermae and Gymnospermae.

In one embodiment, a monocotyledonous plant is used. Monocotyledonousplants belong to the orders of the Alismatales, Arales, Arecales,Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales,Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales,Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plantsbelonging to the class of the Gymnospermae are Cycadales, Ginkgoales,Gnetales, and Pinales. In a particular embodiment, the monocotyledonousplant can be selected from the group consisting of a maize, rice, wheat,barley, and sugarcane.

In another embodiment, a dicotyledonous plant is used, including thosebelonging to the orders of the Aristochiales, Asterales, Batales,Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales,Cornales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales,Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales,Haloragales, Hamamelidales, Middles, Juglandales, Lamiales, Laurales,Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales,Nymphaeales, Papeverales, Piperales, Plantaginales, Plumb aginales,Podostemales, Polemoniales, Polygalales, Polygonales, Primulales,Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales,Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales,Theales, Trochodendrales, Umbellales, Urticales, and Violates. In aparticular embodiment, the dicotyledonous plant can be selected from thegroup consisting of cotton, soybean, pepper, and tomato.

The present invention contemplates the use of endophytic microbialentities derived from plants. It is contemplated that the plants may beagricultural plants. In some embodiments, a cultivar or variety that isof the same family as the plant from which the endophyte is derived isused. In some embodiments, a cultivar or variety that is of the samegenus as the plant from which the endophyte is derived is used. In someembodiments, a cultivar or variety that is of the same species as theancestral plant from which the endophyte is derived is used. In someembodiments, a modern cultivar or variety that is of the same family asthe ancestral plant from which the endophyte is derived is used. Inanother embodiment, a modern cultivar or variety that is of the samegenus as the ancestral plant from which the endophyte is used. In stillanother embodiment, a modern cultivar or variety that is of the samespecies as the ancestral plant from which the endophyte is used.

The methods and compositions of the present invention are preferablyused in plants that are important or interesting for agriculture,horticulture, biomass for the production of biofuel molecules and otherchemicals, and/or forestry. Non-limiting examples include, for instance,Panicum virgatum (switch), Sorghum bicolor (sorghum, sudan), Miscanthusgiganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera(poplar), Zea mays (corn), Glycine max (soybean), Brassica napus(canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryzasativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa),Beta vulgaris (sugarbeet), Pennisetum glaucum (pearl millet), Panicumspp., Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp.,Populus spp., Secale cereale (rye), Salix spp. (willow), Eucalyptus spp.(eucalyptus), Triticosecale spp. (triticum—wheat X rye), Bamboo,Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricinuscommunis (castor), Elaeis guineensis (oil palm), Phoenix dactylifera(date palm), Archontophoenix cunninghamiana (king palm), Syagrusromanzoffiana (queen palm), Linum usitatissimum (flax), Brassica juncea,Manihot esculenta (cassaya), Lycopersicon esculentum (tomato), Lactucasaliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato),Brassica oleracea (broccoli, cauliflower, brusselsprouts), Camelliasinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa),Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus(pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion),Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima(squash), Cucurbita moschata (squash), Spinacea oleracea (spinach),Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanummelongena (eggplant), Papaver somniferum (opium poppy), Papaverorientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabissaliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea,Cinchona officinalis, Coichicum autumnale, Veratrum californica,Digitalis lanata, Digitalis purpurea, Dioscorea spp., Andrographispaniculata, Atropa belladonna, Datura stomonium, Berberis spp.,Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca,Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperziaserrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp.,Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis,Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium,Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata(mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosaspp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia),Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco),Lupinus albus (lupin), Uniola paniculata (oats), Hordeum vulgare(barley), and Lolium spp. (rye).

The present invention contemplates improving an agronomic trait in anagricultural plant by contacting a modern agricultural plant with aformulation comprising an endophyte derived from a plant or an endophyteconserved across diverse species and/or cultivars of agriculturalplants. In one embodiment, the modern agricultural plant is a hybridplant. In another embodiment, the modern agricultural plant is an inbredplant. Non-limiting examples of such hybrid, inbred and geneticallymodified plants are described below. In still another embodiment themodern agricultural plant is a genetically modified plant. The methodsdescribed herein can also be used with genetically modified plants, forexample, to yield additional trait benefits to a plant. In oneembodiment, the modern agricultural plant is a genetically modifiedplant that comprises a transgene that confers in the plant a phenotypeselected from the group consisting of: altered oil content, alteredprotein content, altered seed carbohydrate composition, altered seed oilcomposition, and altered seed protein composition, chemical tolerance,cold tolerance, delayed senescence, disease resistance, droughttolerance, ear weight, growth improvement, health enhancement, heattolerance, herbicide tolerance, herbivore resistance, improved nitrogenfixation, improved nitrogen utilization, improved root architecture,improved water use efficiency, increased biomass, increased root length,increased seed weight, increased shoot length, increased yield,increased yield under water-limited conditions, kernel mass, kernelmoisture content, metal tolerance, number of ears, number of kernels perear, number of pods, nutrition enhancement, pathogen resistance, pestresistance, photosynthetic capability improvement, salinity tolerance,stay-green, vigor improvement, increased dry weight of mature seeds,increased fresh weight of mature seeds, increased number of mature seedsper plant, increased chlorophyll content, increased number of pods perplant, increased length of pods per plant, reduced number of wiltedleaves per plant, reduced number of severely wilted leaves per plant,and increased number of non-wilted leaves per plant, a detectablemodulation in the level of a metabolite, a detectable modulation in thelevel of a transcript, a detectable modulation in the proteome relativeto a reference plant, or any combination thereof.

Plant Element and Endophyte Combinations

It is contemplated that the methods and compositions of the presentinvention may be used to improve any characteristic of any agriculturalplant. The methods described herein can also be used with transgenicplants comprising one or more exogenous transgenes, for example, toyield additional trait benefits conferred by the newly introducedendophytic microbes. Therefore, in one embodiment, a plant element of atransgenic maize, wheat, rice, cotton, canola, alfalfa, or barley plantis contacted with an endophytic microbe.

The presence of the endophyte or other microbes can be detected and itslocalization in or on the host plant (including a plant element thereof)can be determined using a number of different methodologies. Thepresence of the microbe in the embryo or endosperm, as well as itslocalization with respect to the plant cells, can be determined usingmethods known in the art, including immunofluorescence microscopy usingmicrobe specific antibodies, or fluorescence in situ hybridization (see,for example, Amann et al. (2001) Current Opinion in Biotechnology12:231-236, incorporated herein by reference). The presence and quantityof other microbes can be established by the FISH, immunofluorescence andPCR methods using probes that are specific for the microbe.Alternatively, degenerate probes recognizing conserved sequences frommany bacteria and/or fungi can be employed to amplify a region, afterwhich the identity of the microbes present in the tested tissue/cell canbe determined by sequencing.

In some embodiments, the present invention contemplates the use ofendophytes that can confer a beneficial agronomic trait upon the plantelement or resulting plant with which it is associated.

In some cases, the endophytes described herein are capable of movingfrom one tissue type to another. For example, the present invention'sdetection and isolation of endophytes within the mature tissues ofplants after coating on the exterior of a seed demonstrates theirability to move from seed exterior into the vegetative tissues of amaturing plant. Therefore, in one embodiment, the population ofendophytes is capable of moving from the seed exterior into thevegetative tissues of a plant. In one embodiment, the endophyte that iscoated onto the seed of a plant is capable, upon germination of the seedinto a vegetative state, of localizing to a different tissue of theplant. For example, endophytes can be capable of localizing to any oneof the tissues in the plant, including: the root, adventitious root,seminal root, root hair, shoot, leaf, flower, bud, tassel, meristem,pollen, pistil, ovaries, stamen, fruit, stolon, rhizome, nodule, tuber,trichome, guard cells, hydathode, petal, sepal, glume, rachis, vascularcambium, phloem, and xylem. In one embodiment, the endophyte is capableof localizing to the root and/or the root hair of the plant. In anotherembodiment, the endophyte is capable of localizing to the photosynthetictissues, for example, leaves and shoots of the plant. In other cases,the endophyte is localized to the vascular tissues of the plant, forexample, in the xylem and phloem. In still another embodiment, theendophyte is capable of localizing to the reproductive tissues (flower,pollen, pistil, ovaries, stamen, fruit) of the plant. In anotherembodiment, the endophyte is capable of localizing to the root, shoots,leaves and reproductive tissues of the plant. In still anotherembodiment, the endophyte colonizes a fruit or seed tissue of the plant.In still another embodiment, the endophyte is able to colonize the plantsuch that it is present in the surface of the plant (i.e., its presenceis detectably present on the plant exterior, or the episphere of theplant). In still other embodiments, the endophyte is capable oflocalizing to substantially all, or all, tissues of the plant. Incertain embodiments, the endophyte is not localized to the root of aplant. In other cases, the endophyte is not localized to thephotosynthetic tissues of the plant.

In some cases, endophytes are capable of replicating within the hostplant and colonizing the plant.

In another aspect, the present invention provides for combinations ofendophytic microbial entities and plants. The endophytic microbialentities described herein are unique in that they have been isolatedfrom seeds of plants (e.g., an agricultural plant, for example a seed orseedling or an agricultural plant, comprising a population of endophyticmicrobial entities that is heterologously disposed on an exteriorsurface of or within the seed or seedling in an amount effective tocolonize the plant). The combination can further comprise a formulationthat comprises at least one member selected from the group consisting ofan agriculturally compatible carrier, a tackifier, a microbialstabilizer, a fungicide, an antibacterial agent, an herbicide, anematicide, an insecticide, a plant growth regulator, a rodenticide, anda nutrient. In some embodiments, the population of endophytic bacteriaare present in an amount effective to provide a benefit to anagricultural plant derived from an agricultural seed or seedling towhich the formulation is administered.

The population of endophytic microbial entities comprises a nucleic acidsequence that is at least 95%, at least 96%, at least 97% identical, forexample, at least 98%, at least 99%, at least 99.5% identical, 99.8%identical, or 100% identical, to a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 1-3700. In another embodiment, theendophyte comprises a nucleic acid sequence that is at least 99%identical to a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 1-3700. In still another embodiment, the endophytecomprises a nucleic acid sequence that is identical to a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 1-3700.

In some embodiments, disclosed is a seed of an agricultural plantcomprising an exogenous population of an endophyte that is disposed onan exterior surface of or within the plant in an amount effective tocolonize the plant. The population is considered exogenous to the seedif that particular seed does not inherently contain the population ofendophytic microbial entities.

In other cases, the present invention discloses a seed of anagricultural plant comprising a population of endophytic microbialentities that is heterologously disposed on an exterior surface of orwithin the plant in an amount effective to colonize the plant. Forexample, the population of endophytic microbial entities that isdisposed on an exterior surface or within the seed can be an endophytethat may be associated with the mature plant, but is not found on thesurface of or within the seed. Alternatively, the population can befound in the surface of, or within the seed, but at a much lower numberthan is disposed.

As shown in the Examples section below, the endophytic populationsdescribed herein are capable of colonizing the host plant. In certaincases, the endophytic population can be applied to the plant, forexample the plant seed, or by foliar application, and successfulcolonization can be confirmed by detecting the presence of theendophytic microbial population within the plant. For example, afterapplying the endophyte to the seeds, high titers of the endophyte can bedetected in the roots and shoots of the plants that germinate from theseeds. In addition, significant quantities of the endophyte can bedetected in the rhizosphere of the plants. Therefore, in one embodiment,the endophytic microbe population is disposed in an amount effective tocolonize the plant. Colonization of the plant can be detected, forexample, by detecting the presence of the endophytic microbe inside theplant. This can be accomplished by measuring the viability of themicrobe after surface sterilization of the seed or the plant: endophyticcolonization results in an internal localization of the microbe,rendering it resistant to conditions of surface sterilization. Thepresence and quantity of the microbe can also be established using othermeans known in the art, for example, immunofluorescence microscopy usingmicrobe specific antibodies, or fluorescence in situ hybridization (see,for example, Amann et al. (2001) Current Opinion in Biotechnology12:231-236, incorporated herein by reference in its entirety).Alternatively, specific nucleic acid probes recognizing conservedsequences from the endophytic bacterium can be employed to amplify aregion, for example by quantitative PCR, and correlated to CFUs by meansof a standard curve.

In another embodiment, the endophytic microbe is disposed, for example,on the surface of a seed of an agricultural plant, in an amounteffective to be detectable in the mature agricultural plant. In oneembodiment, the endophytic microbe is disposed in an amount effective tobe detectable in an amount of at least about 100 CFU or spores, at leastabout 200 CFU or spores, at least about 300 CFU or spores, at leastabout 500 CFU or spores, at least about 1,000 CFU or spores, at leastabout 3,000 CFU or spores, at least about 10,000 CFU or spores, at leastabout 30,000 CFU or spores, at least about 100,000 CFU or spores, atleast about 10̂6 CFU or spores or more in the mature agricultural plant.

In some cases, the endophytic microbe is capable of colonizingparticular tissue types of the plant. In one embodiment, the endophyticmicrobe is disposed on the seed or seedling in an amount effective to bedetectable within a target tissue of the mature agricultural plantselected from a fruit, a seed, a leaf, or a root, or portion thereof.For example, the endophytic microbe can be detected in an amount of atleast about 100 CFU or spores, at least about 200 CFU or spores, atleast about 300 CFU or spores, at least about 500 CFU or spores, atleast about 1,000 CFU or spores, at least about 3,000 CFU or spores, atleast about 10,000 CFU or spores, at least about 30,000 CFU or spores,at least about 100,000 CFU or spores, at least about 10̂6 CFU or sporesor more, in the target tissue of the mature agricultural plant.

In some cases, the microbes disposed on the seed or seedling can bedetected in the rhizosphere. This may be due to successful colonizationby the endophytic microbe, where certain quantities of the microbe isshed from the root, thereby colonizing the rhizosphere. In some cases,the rhizosphere-localized microbe can secrete compounds (such assiderophores or organic acids) which assist with nutrient acquisition bythe plant. Therefore, in another embodiment, the endophytic microbe isdisposed on the surface of the seed in an amount effective to detectablycolonize the soil environment surrounding the mature agricultural plantwhen compared with a reference agricultural plant. For example, themicrobe can be detected in an amount of at least 100 CFU or spores/g DW,for example, at least 200 CFU or spores/g DW, at least 500 CFU orspores/g DW, at least 1,000 CFU or spores/g DW, at least 3,000 CFU orspores/g DW, at least 10,000 CFU or spores/g DW, at least 30,000 CFU orspores/g DW, at least 100,000 CFU or spores/g DW, at least 300,000 CFUor spores/g DW, or more, in the rhizosphere.

The populations of endophytic microbial entities described herein arealso capable of providing many agronomic benefits to the host plant. Asshown in the Examples section, endophyte-inoculated plants displayincreased seed germination, increased vigor, increased biomass (e.g.,increased root or shoot biomass). Therefore, in one embodiment, thepopulation is disposed on the surface or within a tissue of the seed orseedling in an amount effective to increase the biomass of the plant, ora part or tissue of the plant grown from the seed or seedling.

The increased biomass is useful in the production of commodity productsderived from the plant. Such commodity products include an animal feed,a fish fodder, a cereal product, a processed human-food product, a sugaror an alcohol. Such products may be a fermentation product or afermentable product, one such exemplary product is a biofuel. Theincrease in biomass can occur in a part of the plant (e.g., the roottissue, shoots, leaves, etc.), or can be an increase in overall biomass.Increased biomass production, such an increase meaning at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%when compared with a reference agricultural plant. Such increase inoverall biomass can be under relatively stress-free conditions. In othercases, the increase in biomass can be in plants grown under any numberof abiotic or biotic stresses, including drought stress, salt stress,heat stress, cold stress, low nutrient stress, nematode stress, insectherbivory stress, fungal pathogen stress, bacterial pathogen stress, andviral pathogen stress. In one particular embodiment, the endophyticmicrobial population is disposed in an amount effective to increase rootbiomass by at least 10%, for example, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 75%, at least 100%, ormore, when compared with a reference agricultural plant.

In another embodiment, the endophytic microbial population is disposedon the surface or within a tissue of the seed or seedling in an amounteffective to increase the rate of seed germination when compared with areference agricultural plant. For example, the increase in seedgermination can be at least 2%, at least 3%, at least 4%, at least 5%,at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, atleast 15%, for example, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 75%, at least 100%, or more, whencompared with a reference agricultural plant.

In other cases, the endophytic microbe is disposed on the seed orseedling in an amount effective to increase the average biomass of thefruit or cob from the resulting plant by at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 15%, for example, at least 20%, at least 30%,at least 40%, at least 50%, at least 75%, at least 100% or more, whencompared with a reference agricultural plant.

In some cases, plants are inoculated with endophytes that are isolatedfrom the same species of plant as the plant element of the inoculatedplant. For example, an endophyte that is normally found in one varietyof Zea mays (corn) is associated with a plant element of a plant ofanother variety of Zea mays that in its natural state lacks saidendophyte. In one embodiment, the endophyte is derived from a plant of arelated species of plant as the plant element of the inoculated plant.For example, an endophyte that is normally found in Zea diploperennisIltis et al., (diploperennial teosinte) is applied to a Zea mays (corn),or vice versa. In some cases, plants are inoculated with endophytes thatare heterologous to the plant element of the inoculated plant. In oneembodiment, the endophyte is derived from a plant of another species.For example, an endophyte that is normally found in dicots is applied toa monocot plant (e.g., inoculating corn with a soybean-derivedendophyte), or vice versa. In other cases, the endophyte to beinoculated onto a plant is derived from a related species of the plantthat is being inoculated. In one embodiment, the endophyte is derivedfrom a related taxon, for example, from a related species. The plant ofanother species can be an agricultural plant. In another embodiment, theendophyte is part of a designed composition inoculated into any hostplant element.

As highlighted in the Examples section, plants inoculated with theendophytic microbial population also show an increase in overall plantheight. Therefore, in one embodiment, the present invention provides fora seed comprising an endophytic microbial population which is disposedon the surface or within a tissue of the seed or seedling in an amounteffective to increase the height of the plant. For example, theendophytic microbial population is disposed in an amount effective toresult in an increase in height of the agricultural plant such that isat least 10% greater, for example, at least 20% greater, at least 30%greater, at least 40% greater, at least 50% greater, at least 60%greater, at least 70% greater, at least 80% greater, at least 90%greater, at least 100% greater, at least 125% greater, at least 150%greater or more, when compared with a reference agricultural plant, theplant. Such increase in height can be under relatively stress-freeconditions. In other cases, the increase in height can be in plantsgrown under any number of abiotic or biotic stresses, including droughtstress, salt stress, heat stress, cold stress, low nutrient stress,nematode stress, insect herbivory stress, fungal pathogen stress,bacterial pathogen stress, and viral pathogen stress.

The host plants inoculated with the endophytic microbial population alsoshow dramatic improvements in their ability to utilize water moreefficiently. Water use efficiency is a parameter often correlated withdrought tolerance. Water use efficiency (WUE) is a parameter oftencorrelated with drought tolerance, and is the CO2 assimilation rate perwater transpired by the plant. An increase in biomass at low wateravailability may be due to relatively improved efficiency of growth orreduced water consumption. In selecting traits for improving crops, adecrease in water use, without a change in growth would have particularmerit in an irrigated agricultural system where the water input costswere high. An increase in growth without a corresponding jump in wateruse would have applicability to all agricultural systems. In manyagricultural systems where water supply is not limiting, an increase ingrowth, even if it came at the expense of an increase in water use alsoincreases yield.

When soil water is depleted or if water is not available during periodsof drought, crop yields are restricted. Plant water deficit develops iftranspiration from leaves exceeds the supply of water from the roots.The available water supply is related to the amount of water held in thesoil and the ability of the plant to reach that water with its rootsystem. Transpiration of water from leaves is linked to the fixation ofcarbon dioxide by photosynthesis through the stomata. The two processesare positively correlated so that high carbon dioxide influx throughphotosynthesis is closely linked to water loss by transpiration. Aswater transpires from the leaf, leaf water potential is reduced and thestomata tend to close in a hydraulic process limiting the amount ofphotosynthesis. Since crop yield is dependent on the fixation of carbondioxide in photosynthesis, water uptake and transpiration arecontributing factors to crop yield. Plants which are able to use lesswater to fix the same amount of carbon dioxide or which are able tofunction normally at a lower water potential have the potential toconduct more photosynthesis and thereby to produce more biomass andeconomic yield in many agricultural systems. An increased water useefficiency of the plant relates in some cases to an increasedfruit/kernel size or number.

Therefore, in one embodiment, the plants described herein exhibit anincreased water use efficiency when compared with a referenceagricultural plant grown under the same conditions. For example, theplants grown from the seeds comprising the endophytic microbialpopulation can have at least 5% higher WUE, for example, at least 10%higher, at least 20% higher, at least 30% higher, at least 40% higher,at least 50% higher, at least 60% higher, at least 70% higher, at least80% higher, at least 90% higher, at least 100% higher WUE than areference agricultural plant grown under the same conditions. Such anincrease in WUE can occur under conditions without water deficit, orunder conditions of water deficit, for example, when the soil watercontent is less than or equal to 60% of water saturated soil, forexample, less than or equal to 50%, less than or equal to 40%, less thanor equal to 30%, less than or equal to 20%, less than or equal to 10% ofwater saturated soil on a weight basis.

In a related embodiment, the plant comprising the endophytic microbialpopulation can have at least 10% higher relative water content (RWC),for example, at least 20% higher, at least 30% higher, at least 40%higher, at least 50% higher, at least 60% higher, at least 70% higher,at least 80% higher, at least 90% higher, at least 100% higher RWC thana reference agricultural plant grown under the same conditions.

Many of the microbes described herein are capable of producing the planthormone auxin indole-3-acetic acid (IAA) when grown in culture. Auxinmay play a key role in altering the physiology of the plant, includingthe extent of root growth. Therefore, in another embodiment, theendophytic microbial population is disposed on the surface or within atissue of the seed or seedling in an amount effective to detectablyinduce production of auxin in the agricultural plant. For example, theincrease in auxin production can be at least 2%, at least 3%, at least4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, atleast 10%, at least 15%, for example, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 75%, at least 100%, ormore, when compared with a reference agricultural plant. In oneembodiment, the increased auxin production can be detected in a tissuetype selected from the group consisting of the root, shoot, leaves, andflowers.

In another embodiment, the endophytic population of the presentinvention can cause a detectable modulation in the amount of ametabolite in the plant or part of the plant. Such modulation can bedetected, for example, by measuring the levels of a given metabolite andcomparing with the levels of the metabolite in a reference agriculturalplant grown under the same conditions.

Formulations for Agricultural Use

The present invention contemplates a synthetic combination of a plantelement that is associated with an endophyte to confer an improved traitof agronomic importance to the host plant, or an improved agronomictrait potential to a plant element associated with the endophyte, thatupon and after germination will confer said benefit to the resultanthost plant.

In some embodiments, the plant element is associated with an endophyteon its surface. Such association is contemplated to be via a mechanismselected from the group consisting of: spraying, immersion, coating,encapsulating, dusting, dripping, aerosolizing.

In some embodiments, the plant element is a leaf, and the syntheticcombination is formulated for application as a foliar treatment.

In some embodiments, the plant element is a seed, and the syntheticcombination is formulated for application as a seed coating.

In some embodiments, the plant element is a root, and the syntheticcombination is formulated for application as a root treatment.

In certain embodiments, the plant element becomes associated with theendophyte(s) through delayed exposure. For example, the soil in which aplant element is to be introduced is first treated with a compositioncomprising the endophyte or combination of endophytes. In anotherexample, the area around the plant or plant element is exposed to aformulation comprising the endophyte(s), and the plant element becomessubsequently associated with the endophyte(s) due to movement of soil,air, water, insects, mammals, human intervention, or other methods.

The plant element can be obtained from any agricultural plant. In oneembodiment, the plant element of the first plant is from amonocotyledonous plant. For example, the plant element of the firstplant is from a cereal plant. The plant element of the first plant canbe selected from the group consisting of a maize seed, a wheat seed, abarley seed, a rice seed, a sugarcane seed, a maize root, a wheat root,a barley root, a sugarcane root, a rice root, a maize leaf, a wheatleaf, a barley leaf, a sugarcane leaf, or a rice leaf. In an alternativeembodiment, the plant element of the first plant is from adicotyledonous plant. The plant element of the first plant can beselected from the group consisting of a cotton seed, a tomato seed, acanola seed, a pepper seed, a soybean seed, a cotton root, a tomatoroot, a canola root, a pepper root, a soybean root, a cotton leaf, atomato leaf, a canola leaf, a pepper leaf, or a soybean leaf. In stillanother embodiment, the plant element of the first plant can be from agenetically modified plant. In another embodiment, the plant element ofthe first plant can be a hybrid plant element.

The synthetic combination can comprise a plant element of the firstplant which is surface-sterilized prior to combining with theendophytes. Such pre-treatment prior to coating the seed with endophytesremoves the presence of other microbes which may interfere with theoptimal colonization, growth and/or function of the endophyte. Surfacesterilization of seeds can be accomplished without killing the seeds asdescribed herein.

The endophyte populations described herein are intended to be useful inthe improvement of agricultural plants, and as such, may be formulatedwith other compositions as part of an agriculturally compatible carrier.It is contemplated that such carriers can include, but not be limitedto: seed treatment, root treatment, foliar treatment, soil treatment.The carrier composition with the endophyte populations, may be preparedfor agricultural application as a liquid, a solid, or a gas formulation.Application to the plant may be achieved, for example, as a powder forsurface deposition onto plant leaves, as a spray to the whole plant orselected plant element, as part of a drip to the soil or the roots, oras a coating onto the seed prior to planting. Such examples are meant tobe illustrative and not limiting to the scope of the invention.

In some embodiments, the present invention contemplates plant elementscomprising a endophytic microbial population, and further comprising aformulation. The formulation useful for these embodiments generallycomprises at least one member selected from the group consisting of anagriculturally compatible carrier, a tackifier, a microbial stabilizer,a fungicide, an antibacterial agent, an herbicide, a nematicide, aninsecticide, a plant growth regulator, a rodenticide, and a nutrient.

In some cases, the endophytic population is mixed with an agriculturallycompatible carrier. The carrier can be a solid carrier or liquidcarrier. The carrier may be any one or more of a number of carriers thatconfer a variety of properties, such as increased stability,wettability, or dispersability. Wetting agents such as natural orsynthetic surfactants, which can be nonionic or ionic surfactants, or acombination thereof can be included in a composition of the invention.Water-in-oil emulsions can also be used to formulate a composition thatincludes the endophytic population of the present invention (see, forexample, U.S. Pat. No. 7,485,451, which is incorporated herein byreference in its entirety). Suitable formulations that may be preparedinclude wettable powders, granules, gels, agar strips or pellets,thickeners, and the like, microencapsulated particles, and the like,liquids such as aqueous flowables, aqueous suspensions, water-in-oilemulsions, etc. The formulation may include grain or legume products,for example, ground grain or beans, broth or flour derived from grain orbeans, starch, sugar, or oil.

In some embodiments, the agricultural carrier may be soil or plantgrowth medium. Other agricultural carriers that may be used includefertilizers, plant-based oils, humectants, or combinations thereof.Alternatively, the agricultural carrier may be a solid, such asdiatomaceous earth, loam, silica, alginate, clay, bentonite,vermiculite, seed cases, other plant and animal products, orcombinations, including granules, pellets, or suspensions. Mixtures ofany of the aforementioned ingredients are also contemplated as carriers,such as but not limited to, pesta (flour and kaolin clay), agar orflour-based pellets in loam, sand, or clay, etc. Formulations mayinclude food sources for the cultured organisms, such as barley, rice,or other biological materials such as seed, leaf, root, plant elements,sugar cane bagasse, hulls or stalks from grain processing, ground plantmaterial or wood from building site refuse, sawdust or small fibers fromrecycling of paper, fabric, or wood. Other suitable formulations will beknown to those skilled in the art.

In one embodiment, the formulation can comprise a tackifier or adherent.Such agents are useful for combining the microbial population of theinvention with carriers that can contain other compounds (e.g., controlagents that are not biologic), to yield a coating composition. Suchcompositions help create coatings around the plant or plant element tomaintain contact between the microbe and other agents with the plant orplant part. In one embodiment, adherents are selected from the groupconsisting of: alginate, gums, starches, lecithins, formononetin,polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinylacetate, cephalins, Gum Arabic, Xanthan Gum, Mineral Oil, PolyethyleneGlycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, MethylCellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate,Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate, GellanGum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum Ghatti, andpolyoxyethylene-polyoxybutylene block copolymers. Other examples ofadherent compositions that can be used in the synthetic preparationinclude those described in EP 0818135, CA 1229497, WO 2013090628, EP0192342, WO 2008103422 and CA 1041788, each of which is incorporatedherein by reference in its entirety.

The formulation can also contain a surfactant. Non-limiting examples ofsurfactants include nitrogen-surfactant blends such as Prefer 28(Cenex), Surf-N (US), Inhance (Brandt), P-28 (Wilfarm) and Patrol(Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP),Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); andorgano-silicone surfactants include Silwet L77 (UAP), Silikin (Terra),Dyne-Amic (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) andCentury (Precision). In one embodiment, the surfactant is present at aconcentration of between 0.01% v/v to 10% v/v. In another embodiment,the surfactant is present at a concentration of between 0.1% v/v to 1%v/v.

In certain cases, the formulation includes a microbial stabilizer. Suchan agent can include a desiccant. As used herein, a “desiccant” caninclude any compound or mixture of compounds that can be classified as adesiccant regardless of whether the compound or compounds are used insuch concentrations that they in fact have a desiccating effect on theliquid inoculant. Such desiccants are ideally compatible with theendophytic population used, and should promote the ability of themicrobial population to survive application on the plant elements and tosurvive desiccation. Examples of suitable desiccants include one or moreof trehalose, sucrose, glycerol, and Methylene glycol. Other suitabledesiccants include, but are not limited to, non-reducing sugars andsugar alcohols (e.g., mannitol or sorbitol). The amount of desiccantintroduced into the formulation can range from about 5% to about 50% byweight/volume, for example, between about 10% to about 40%, betweenabout 15% and about 35%, or between about 20% and about 30%.

In some cases, it is advantageous for the formulation to contain agentssuch as a fungicide, an antibacterial agent, an herbicide, a nematicide,an insecticide, a plant growth regulator, a rodenticide, and a nutrient.Such agents are ideally compatible with the agricultural plant elementor seedling onto which the formulation is applied (e.g., it should notbe deleterious to the growth or health of the plant). Furthermore, theagent is ideally one which does not cause safety concerns for human,animal or industrial use (e.g., no safety issues, or the compound issufficiently labile that the commodity plant product derived from theplant contains negligible amounts of the compound).

In the liquid form, for example, solutions or suspensions, theendophytic microbial populations of the present invention can be mixedor suspended in aqueous solutions. Suitable liquid diluents or carriersinclude aqueous solutions, petroleum distillates, or other liquidcarriers.

Solid compositions can be prepared by dispersing the endophyticmicrobial populations of the invention in and on an appropriatelydivided solid carrier, such as peat, wheat, bran, vermiculite, clay,talc, bentonite, diatomaceous earth, fuller's earth, pasteurized soil,and the like. When such formulations are used as wettable powders,biologically compatible dispersing agents such as non-ionic, anionic,amphoteric, or cationic dispersing and emulsifying agents can be used.

The solid carriers used upon formulation include, for example, mineralcarriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite,diatomaceous earth, acid white soil, vermiculite, and pearlite, andinorganic salts such as ammonium sulfate, ammonium phosphate, ammoniumnitrate, urea, ammonium chloride, and calcium carbonate. Also, organicfine powders such as wheat flour, wheat bran, and rice bran may be used.The liquid carriers include vegetable oils such as soybean oil andcottonseed oil, glycerol, ethylene glycol, polyethylene glycol,propylene glycol, polypropylene glycol, etc.

In one particular embodiment, the formulation is ideally suited forcoating of the endophytic microbial population onto plant elements. Theendophytic microbial populations described in the present invention arecapable of conferring many agronomic benefits to the host plants. Theability to confer such benefits by coating the endophytic microbialpopulations on the surface of plant elements has many potentialadvantages, particularly when used in a commercial (agricultural) scale.

The endophytic microbial populations herein can be combined with one ormore of the agents described above to yield a formulation suitable forcombining with an agricultural plant element or seedling. The endophyticmicrobial population can be obtained from growth in culture, forexample, using a synthetic growth medium. In addition, the microbe canbe cultured on solid media, for example on petri dishes, scraped off andsuspended into the preparation. Microbes at different growth phases canbe used. For example, microbes at lag phase, early-log phase, mid-logphase, late-log phase, stationary phase, early death phase, or deathphase can be used.

The formulations comprising the endophytic microbial population of thepresent invention typically contains between about 0.1 to 95% by weight,for example, between about 1% and 90%, between about 3% and 75%, betweenabout 5% and 60%, between about 10% and 50% in wet weight of theendophytic population of the present invention. It is preferred that theformulation contains at least about 10̂2 per ml of formulation, at leastabout 10̂3 per ml of formulation, for example, at least about 10̂4, atleast about 10̂5, at least about 10̂6, at least about 10̂7 CFU or spores,at least about 10̂8 CFU or spores per ml of formulation.

As described above, in certain embodiments, the present inventioncontemplates the use of endophytic bacteria and/or fungi that areheterologously disposed on the plant, for example, the plant element. Incertain cases, the agricultural plant may contain bacteria that aresubstantially similar to, or even genetically indistinguishable from,the bacteria that are being applied to the plant. It is noted that, inmany cases, the bacteria that are being applied is substantiallydifferent from the bacteria already present in several significant ways.First, the bacteria that are being applied to the agricultural planthave been adapted to culture, or adapted to be able to grow on growthmedia in isolation from the plant. Second, in many cases, the bacteriathat are being applied are derived from a clonal origin, rather thanfrom a heterologous origin and, as such, can be distinguished from thebacteria that are already present in the agricultural plant by theclonal similarity. For example, where a microbe that has been inoculatedby a plant is also present in the plant (for example, in a differenttissue or portion of the plant), or where the introduced microbe issufficiently similar to a microbe that is present in some of the plants(or portion of the plant, including plant elements), it is stillpossible to distinguish between the inoculated microbe and the nativemicrobe by distinguishing between the two microbe types on the basis oftheir epigenetic status (e.g., the bacteria that are applied, as well astheir progeny, would be expected to have a much more uniform and similarpattern of cytosine methylation of its genome, with respect to theextent and/or location of methylation).

Endophytes Compatible with Agrichemicals

In certain embodiments, the endophyte is selected on the basis of itscompatibility with commonly used agrichemicals. As mentioned earlier,plants, particularly agricultural plants, can be treated with a vastarray of agrichemicals, including fungicides, biocides (anti-complexagents), herbicides, insecticides, nematicides, rodenticides,fertilizers, and other agents.

In some cases, it can be important for the endophyte to be compatiblewith agrichemicals, particularly those with anticomplex properties, inorder to persist in the plant although, as mentioned earlier, there aremany such anticomplex agents that do not penetrate the plant, at leastat a concentration sufficient to interfere with the endophyte.Therefore, where a systemic anticomplex agent is used in the plant,compatibility of the endophyte to be inoculated with such agents will bean important criterion.

Fungicides.

In one embodiment, the control agent is a fungicide. As used herein, afungicide is any compound or agent (whether chemical or biological) thatcan either inhibit the growth of a fungus or kill a fungus. In thatsense, a “fungicide”, as used herein, encompasses compounds that may befungistatic or fungicidal. As used herein, the fungicide can be aprotectant, or agents that are effective predominantly on the seedsurface, providing protection against seed surface-borne pathogens andproviding some level of control of soil-borne pathogens. Non-limitingexamples of protectant fungicides include captan, maneb, thiram, orfludioxonil.

The fungicide can be a systemic fungicide, which can be absorbed intothe emerging seedling and inhibit or kill the fungus inside host planttissues. Systemic fungicides used for seed treatment include, but arenot limited to the following: azoxystrobin, carboxin, mefenoxam,metalaxyl, thiabendazole, trifloxystrobin, and various triazolefungicides, including difenoconazole, ipconazole, tebuconazole, andtriticonazole. Mefenoxam and metalaxyl are primarily used to target thewater mold fungi Pythium and Phytophthora. Some fungicides are preferredover others, depending on the plant species, either because of subtledifferences in sensitivity of the pathogenic fungal species, or becauseof the differences in the fungicide distribution or sensitivity of theplants.

A fungicide can be a biological control agent, such as a bacterium orfungus. Such organisms may be parasitic to the pathogenic fungi, orsecrete toxins or other substances which can kill or otherwise preventthe growth of fungi. Any type of fungicide, particularly ones that arecommonly used on plants, can be used as a control agent in a seedcomposition.

Antibacterial Agents.

In some cases, the seed coating composition comprises a control agentwhich has antibacterial properties. In one embodiment, the control agentwith antibacterial properties is selected from the compounds describedherein elsewhere. In another embodiment, the compound is Streptomycin,oxytetracycline, oxolinic acid, or gentamicin.

Plant Growth Regulators.

The seed coat composition can further comprise a plant growth regulator.In one embodiment, the plant growth regulator is selected from the groupconsisting of: Abscisic acid, amidochlor, ancymidol,6-benzylaminopurine, brassinolide, butralin, chlormequat (chlormequatchloride), choline chloride, cyclanilide, daminozide, dikegulac,dimethipin, 2,6-dimethylpuridine, ethephon, flumetralin, flurprimidol,fluthiacet, forchlorfenuron, gibberellic acid, inabenfide,indole-3-acetic acid, maleic hydrazide, mefluidide, mepiquat (mepiquatchloride), naphthaleneacetic acid, N-6-benzyladenine, paclobutrazol,prohexadione (prohexadione-calcium), prohydrojasmon, thidiazuron,triapenthenol, tributyl phosphorotrithioate, 2,3,5-tri-iodobenzoic acid,trinexapac-ethyl and uniconazole. Other examples of antibacterialcompounds which can be used as part of a seed coating compositioninclude those based on dichlorophene and benzylalcohol hemi formal(Proxel® from ICI or Acticide® RS from Thor Chemie and Kathon® MK fromRohm & Haas) and isothiazolinone derivatives such asalkylisothiazolinones and benzisothiazolinones (Acticide® MBS from ThorChemie). Other plant growth regulators that can be incorporated seedcoating compositions are described in US 2012/0108431, which isincorporated by reference in its entirety.

Nematicides.

Preferred nematode-antagonistic biocontrol agents include ARF18;Arthrobotrys spp.; Chaetomium spp.; Cylindrocarpon spp.; Exophilia spp.;Fusarium spp.; Gliocladium spp.; Hirsutella spp.; Lecanicillium spp.;Monacrosporium spp.; Myrothecium spp.; Neocosmospora spp.; Paecilomycesspp.; Pochonia spp.; Stagonospora spp.; vesicular-arbuscular mycorrhizalfungi, Burkholderia spp.; Pasteuria spp., Brevibacillus spp.;Pseudomonas spp.; and Rhizobacteria. Particularly preferrednematode-antagonistic biocontrol agents include ARF18, Arthrobotrysoligospora, Arthrobotrys dactyloides, Chaetomium globosum,Cylindrocarpon heteronema, Exophilia jeanselmei, Exophilia pisciphila,Fusarium aspergilus, Fusarium solani, Gliocladium catenulatum,Gliocladium roseum, Gliocladium vixens, Hirsutella rhossiliensis,Hirsutella minnesotensis, Lecanicillium lecanii, Monacrosporiumdrechsleri, Monacrosporium gephyropagum, Myrotehcium verrucaria,Neocosmospora vasinfecta, Paecilomyces lilacinus, Pochoniachlamydosporia, Stagonospora heteroderae, Stagonospora phaseoli,vesicular-arbuscular mycorrhizal fungi, Burkholderia cepacia, Pasteuriapenetrans, Pasteuria thornei, Pasteuria nishizawae, Pasteuria ramosa,Pastrueia usage, Brevibacillus laterosporus strain G4, Pseudomonasfluorescens and Rhizobacteria.

Nutrients.

In another embodiment, the seed coating composition can comprise anutrient. The nutrient can be selected from the group consisting of anitrogen fertilizer including, but not limited to Urea, Ammoniumnitrate, Ammonium sulfate, Non-pressure nitrogen solutions, Aquaammonia, Anhydrous ammonia, Ammonium thiosulfate, Sulfur-coated urea,Urea-formaldehydes, IBDU, Polymer-coated urea, Calcium nitrate,Ureaform, and Methylene urea, phosphorous fertilizers such as Diammoniumphosphate, Monoammonium phosphate, Ammonium polyphosphate, Concentratedsuperphosphate and Triple superphosphate, and potassium fertilizers suchas Potassium chloride, Potassium sulfate, Potassium-magnesium sulfate,Potassium nitrate. Such compositions can exist as free salts or ionswithin the seed coat composition. Alternatively, nutrients/fertilizerscan be complexed or chelated to provide sustained release over time.

Rodenticides.

Rodents such as mice and rats cause considerable economical damage byeating and soiling planted or stored seeds. Moreover, mice and ratstransmit a large number of infectious diseases such as plague, typhoid,leptospirosis, trichinosis and salmonellosis. Anticoagulants such ascoumarin and indandione derivatives play an important role in thecontrol of rodents. These active ingredients are simple to handle,relatively harmless to humans and have the advantage that, as the resultof the delayed onset of the activity, the animals being controlledidentify no connection with the bait that they have ingested, thereforedo not avoid it. This is an important aspect in particular in socialanimals such as rats, where individuals act as tasters. In oneembodiment, the seed coating composition comprises a rodenticideselected from the group of substances consisting of2-isovalerylindan-1,3-dione, 4-(quinoxalin-2-ylamino)benzenesulfonamide,alpha-chlorohydrin, aluminum phosphide, antu, arsenous oxide, bariumcarbonate, bisthiosemi, brodifacoum, bromadiolone, bromethalin, calciumcyanide, chloralose, chlorophacinone, cholecalciferol, coumachlor,coumafuryl, coumatetralyl, crimidine, difenacoum, difethialone,diphacinone, ergocalciferol, flocoumafen, fluoroacetamide, flupropadine,flupropadine hydrochloride, hydrogen cyanide, iodomethane, lindane,magnesium phosphide, methyl bromide, norbormide, phosacetim, phosphine,phosphorus, pindone, potassium arsenite, pyrinuron, scilliroside, sodiumarsenite, sodium cyanide, sodium fluoroacetate, strychnine, thalliumsulfate, warfarin and zinc phosphide.

Compatibility

In one embodiment, natural isolates of endophytes that are compatiblewith agrichemicals can be used to inoculate the plants according to themethods described herein. For example, endophytes that are compatiblewith agriculturally employed anticomplex agents can be isolated byplating a culture of endophytes on a petri dish comprising an effectiveconcentration of the anticomplex agent, and isolating colonies ofendophytes that are compatible with the anticomplex agent. In anotherembodiment, an endophyte that is compatible with an anticomplex agent isused for the methods described herein.

Bactericide-compatible endophyte can also be isolated by selection onliquid medium. The culture of endophytes can be plated on petri disheswithout any forms of mutagenesis; alternatively, endophytes can bemutagenized using any means known in the art. For example, endophytecultures can be exposed to UV light, gamma-irradiation, or chemicalmutagens such as ethylmethanesulfonate (EMS) prior to selection onfungicide comprising media. Finally, where the mechanism of action of aparticular bactericide is known, the target gene can be specificallymutated (either by gene deletion, gene replacement, site-directedmutagenesis, etc.) to generate an endophyte that is resilient againstthat particular chemical. It is noted that the above-described methodscan be used to isolate endophytes that are compatible with bothbacteriostatic and bactericidal compounds.

It will also be appreciated by one skilled in the art that a plant maybe exposed to multiple types of anticomplex compounds, eithersimultaneously or in succession, for example at different stages ofplant growth. Where the target plant is likely to be exposed to multipleanticomplex agents, an endophyte that is compatible with many or all ofthese agrichemicals can be used to inoculate the plant. An endophytethat is compatible with several agents can be isolated, for example, byserial selection. An endophyte that is compatible with the first agentcan be isolated as described above (with or without prior mutagenesis).A culture of the resulting endophyte can then be selected for theability to grow on liquid or solid media comprising the second agent(again, with or without prior mutagenesis). Colonies isolated from thesecond selection are then tested to confirm its compatibility to bothagents.

Likewise, endophytes that are compatible to biocides (includingherbicides such as glyphosate or anticomplex compounds, whetherbacteriostatic or bactericidal) that are agriculturally employed can beisolated using methods similar to those described for isolatingcompatible endophytes. In one embodiment, mutagenesis of the endophytepopulation can be performed prior to selection with an anticomplexagent. In another embodiment, selection is performed on the endophytepopulation without prior mutagenesis. In still another embodiment,serial selection is performed on an endophyte: the endophyte is firstselected for compatibility to a first anticomplex agent. The isolatedcompatible endophyte is then cultured and selected for compatibility tothe second anticomplex agent. Any colony thus isolated is tested forcompatibility to each, or both anticomplex agents to confirmcompatibility with these two agents.

Compatibility with an antimicrobial agent can be determined by a numberof means known in the art, including the comparison of the minimalinhibitory concentration (MIC) of the unmodified and modifiedendophytes. Therefore, in one embodiment, the present inventiondiscloses an isolated modified endophyte, wherein the endophyte ismodified such that it exhibits at least 3 fold greater, for example, atleast 5 fold greater, at least 10 fold greater, at least 20 foldgreater, at least 30 fold greater or more MIC to an antimicrobial agentwhen compared with the unmodified endophyte.

In one particular aspect, disclosed herein are endophytes with enhancedcompatibility to the herbicide glyphosate. In one embodiment, theendophyte has a doubling time in growth medium comprising at least 1 mMglyphosate, for example, at least 2 mM glyphosate, at least 5 mMglyphosate, at least 10 mM glyphosate, at least 15 mM glyphosate ormore, that is no more than 250%, for example, no more than 200%, no morethan 175%, no more than 150%, or no more than 125%, of the doubling timeof the endophyte in the same growth medium comprising no glyphosate. Inone particular embodiment, the endophyte has a doubling time in growthmedium comprising 5 mM glyphosate that is no more than 150% the doublingtime of the endophyte in the same growth medium comprising noglyphosate.

In another embodiment, the endophyte has a doubling time in a planttissue comprising at least 10 ppm glyphosate, for example, at least 15ppm glyphosate, at least 20 ppm glyphosate, at least 30 ppm glyphosate,at least 40 ppm glyphosate or more, that is no more than 250%, forexample, no more than 200%, no more than 175%, no more than 150%, or nomore than 125%, of the doubling time of the endophyte in a referenceplant tissue comprising no glyphosate. In one particular embodiment, theendophyte has a doubling time in a plant tissue comprising 40 ppmglyphosate that is no more than 150% the doubling time of the endophytein a reference plant tissue comprising no glyphosate.

The selection process described above can be repeated to identifyisolates of endophytes that are compatible with a multitude of agents.

Candidate isolates can be tested to ensure that the selection foragrichemical compatibility did not result in loss of a desiredbioactivity. Isolates of endophytes that are compatible with commonlyemployed agents can be selected as described above. The resultingcompatible endophyte can be compared with the parental endophyte onplants in its ability to promote germination.

The agrichemical compatible endophytes generated as described above canbe detected in samples. For example, where a transgene was introduced torender the endophyte compatible with the agrichemical(s), the transgenecan be used as a target gene for amplification and detection by PCR. Inaddition, where point mutations or deletions to a portion of a specificgene or a number of genes results in compatibility with theagrichemical(s), the unique point mutations can likewise be detected byPCR or other means known in the art. Such methods allow the detection ofthe endophyte even if it is no longer viable. Thus, commodity plantproducts produced using the agrichemical compatible endophytes describedherein can readily be identified by employing these and related methodsof nucleic acid detection.

Beneficial Attributes of Synthetic Combinations of Plant Elements andEndophytes

Improved Attributes Conferred by Endophytes.

The present invention contemplates the establishment of a symbiont in aplant element. In one embodiment, endophyte association results in adetectable change to the plant element, in particular the seed or thewhole plant. The detectable change can be an improvement in a number ofagronomic traits (e.g., improved general health, increased response tobiotic or abiotic stresses, or enhanced properties of the plant or aplant element, including fruits and grains). Alternatively, thedetectable change can be a physiological or biological change that canbe measured by methods known in the art. The detectable changes aredescribed in more detail in the sections below. As used herein, anendophyte is considered to have conferred an improved agricultural traitwhether or not the improved trait arose from the plant, the endophyte,or the concerted action between the plant and endophyte. Therefore, forexample, whether a beneficial hormone or chemical is produced by theplant or the endophyte, for purposes of the present invention, theendophyte will be considered to have conferred an improved agronomictrait upon the host plant.

In some aspects, provided herein, are methods for producing a seed of aplant with a heritably altered trait. The trait of the plant can bealtered without known genetic modification of the plant genome, andcomprises the following steps. First, a preparation of an isolatedendophyte that is heterologous to the seed of the plant is provided, andoptionally processed to produce an endophyte formulation. The endophyteformulation is then contacted with the plant. The plants are thenallowed to go to seed, and the seeds are collected.

Improved General Health.

Also described herein are plants, and fields of plants, that areassociated with beneficial endophytes, such that the overall fitness,productivity or health of the plant or a portion thereof, is maintained,increased and/or improved over a period of time. Improvement in overallplant health can be assessed using numerous physiological parametersincluding, but not limited to, height, overall biomass, root and/orshoot biomass, seed germination, seedling survival, photosyntheticefficiency, transpiration rate, seed/fruit number or mass, plant grainor fruit yield, leaf chlorophyll content, photosynthetic rate, rootlength, or any combination thereof. Improved plant health, or improvedfield health, can also be demonstrated through improved resistance orresponse to a given stress, either biotic or abiotic stress, or acombination of one or more abiotic stresses, as provided herein.

Other Abiotic Stresses.

Disclosed herein are endophyte-associated plants with increasedresistance to an abiotic stress. Exemplary abiotic stresses include, butare not limited to:

Drought and Heat Tolerance.

In some cases, a plant resulting from seeds or other plant componentstreated with an endophyte can exhibit a physiological change, such as acompensation of the stress-induced reduction in photosynthetic activity(expressed, for example, as ΔFv/Fm) after exposure to heat shock ordrought conditions as compared to a corresponding control, geneticallyidentical plant that does not contain the endophytes grown in the sameconditions. In some cases, the endophyte-associated plant as disclosedherein can exhibit an increased change in photosynthetic activityΔFv(ΔFv/Fm) after heat-shock or drought stress treatment, for example 1,2, 3, 4, 5, 6, 7 days or more after the heat-shock or drought stresstreatment, or until photosynthesis ceases, as compared withcorresponding control plant of similar developmental stage but notcomprising endophytes. For example, a plant having an endophyte able toconfer heat and/or drought-tolerance can exhibit a ΔFv/Fm of from about0.1 to about 0.8 after exposure to heat-shock or drought stress or aΔFv/Fm range of from about 0.03 to about 0.8 under one day, or 1, 2, 3,4, 5, 6, 7, or over 7 days post heat-shock or drought stress treatment,or until photosynthesis ceases. In some embodiments, stress-inducedreductions in photosynthetic activity can be compensated by at leastabout 0.25% (for example, at least about 0.5%, at least about 1%, atleast about 2%, at least about 3, at least about 5%, at least about 8%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 75%, at least about 80%, at leastabout 80%, at least about 90%, at least about 95%, at least about 99% orat least 100%) as compared to the photosynthetic activity decrease in acorresponding reference agricultural plant following heat shockconditions. Significance of the difference between endophyte-associatedand reference agricultural plants can be established upon demonstratingstatistical significance, for example at p<0.05 with an appropriateparametric or non-parametric statistic, e.g., Chi-square test, Student'st-test, Mann-Whitney test, or F-test based on the assumption or knownfacts that the endophyte-associated plant and reference agriculturalplant have identical or near identical genomes (isoline comparison).

In some embodiments, the plants comprise endophytes able to increaseheat and/or drought-tolerance in sufficient quantity, such thatincreased growth or improved recovery from wilting under conditions ofheat or drought stress is observed. For example, an endophyte populationdescribed herein can be present in sufficient quantity in a plant,resulting in increased growth as compared to a plant that does notcontain endophytes, when grown under drought conditions or heat shockconditions, or following such conditions. Increased heat and/or droughttolerance can be assessed with physiological parameters including, butnot limited to, increased height, overall biomass, root and/or shootbiomass, seed germination, seedling survival, photosynthetic efficiency,transpiration rate, seed/fruit number or mass, plant grain or fruityield, leaf chlorophyll content, photosynthetic rate, root length, wiltrecovery, turgor pressure, or any combination thereof, as compared to areference agricultural plant grown under similar conditions.

In various embodiments, endophytes introduced into altered seedmicrobiota can confer in the resulting plant thermal tolerance,herbicide tolerance, drought resistance, insect resistance, fungusresistance, virus resistance, bacteria resistance, male sterility, coldtolerance, salt tolerance, increased yield, enhanced nutrient useefficiency, increased nitrogen use efficiency, increased proteincontent, increased fermentable carbohydrate content, reduced lignincontent, increased antioxidant content, enhanced water use efficiency,increased vigor, increased germination efficiency, earlier or increasedflowering, increased biomass, altered root-to-shoot biomass ratio,enhanced soil water retention, or a combination thereof. A differencebetween the endophyte-associated plant and a reference agriculturalplant can also be measured using other methods known in the art (see,for example, Haake et al. (2002) Plant Physiol. 130: 639-648,incorporated herein by reference in its entirety)

Salt Stress.

In other embodiments, endophytes able to confer increased tolerance tosalinity stress can be introduced into plants. The resulting plantscomprising endophytes can exhibit increased resistance to salt stress,whether measured in terms of survival under saline conditions, oroverall growth during, or following salt stress. The physiologicalparameters of plant health recited above, including height, overallbiomass, root and/or shoot biomass, seed germination, seedling survival,photosynthetic efficiency, transpiration rate, seed/fruit number ormass, plant grain or fruit yield, leaf chlorophyll content,photosynthetic rate, root length, or any combination thereof, can beused to measure growth, and compared with the growth rate of referenceagricultural plants (e.g., isogenic plants without the endophytes) grownunder identical conditions.

In other instances, endophyte-associated plants and referenceagricultural plants can be grown in soil or growth media comprisingdifferent concentration of sodium to establish the inhibitoryconcentration of sodium (expressed, for example, as the concentration inwhich growth of the plant is inhibited by 50% when compared with plantsgrown under no sodium stress). Therefore, in another embodiment, a plantresulting from seeds comprising an endophyte able to confer salttolerance described herein exhibits an increase in the inhibitory sodiumconcentration by at least 10 mM, for example at least 15 mM, at least 20mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, atleast 70 mM, at least 80 mM, at least 90 mM, at least 100 mM or more,when compared with the reference agricultural plants.

High Metal Content.

Plants are sessile organisms and therefore must contend with theenvironment in which they are placed. Plants have adapted manymechanisms to deal with chemicals and substances that may be deleteriousto their health. Heavy metals in particular represent a class of toxinsthat are highly relevant for plant growth and agriculture, because manyof them are associated with fertilizers and sewage sludge used to amendsoils and can accumulate to toxic levels in agricultural fields(Mortvedt 1996, Fertilizer Res. 43:55-61; Kidd et al. 2007, Chemosphere66:1458-1467, incorporated herein by reference in their entirety).Therefore, for agricultural purposes, it is important to have plantsthat are able to tolerate soils comprising elevated levels of toxicheavy metals. Plants cope with toxic levels of heavy metals (forexample, nickel, cadmium, lead, mercury, arsenic, or aluminum) in thesoil by excretion and internal sequestration (Choi et al. 2001, Planta213:45-50; Kumar et al. 1995, Environ. Sci. Technol. 29:1232-1238,incorporated herein by reference in its entirety)). Endophytes that areable to confer increased heavy metal tolerance may do so by enhancingsequestration of the metal in certain compartments away from the seed orfruit and/or by supplementing other nutrients necessary to remediate thestress (Burd et al. 2000, Can. J. Microbiol. 46:237-245; Rajkumar et al.2009, Chemosphere 77:153-160, incorporated herein by reference in theirentirety). Use of such endophytes in a plant would allow the developmentof novel plant-endophyte combinations for purposes of environmentalremediation (also known as phytoremediation). Therefore, in oneembodiment, the plant comprising endophytes shows increased metaltolerance as compared to a reference agricultural plant grown under thesame heavy metal concentration in the soil.

Alternatively, the inhibitory concentration of the heavy metal can bedetermined for endophyte-associated plant and compared with a referenceagricultural plant under the same conditions. Therefore, in oneembodiment, the plants resulting from seeds comprising an endophyte ableto confer heavy metal tolerance described herein exhibit an increase inthe inhibitory sodium concentration by at least 0.1 mM, for example atleast 0.3 mM, at least 0.5 mM, at least 1 mM, at least 2 mM, at least 5mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 30 mM, atleast 50 mM or more, when compared with the reference agriculturalplants.

Finally, plants inoculated with endophytes that are able to conferincreased metal tolerance exhibit an increase in overall metal excretionby at least 10%, for example at least 15%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 75%, at least 100%,at least 150%, at least 200%, at least 300% or more, when compared withuninoculated plants grown under the same conditions.

Low Nutrient Stress.

Endophytes described herein may also confer to the plant an increasedability to grow in nutrient limiting conditions, for example bysolubilizing or otherwise making available to the plants macronutrientsor micronutrients that are complexed, insoluble, or otherwise in anunavailable form. In one embodiment, a plant is inoculated with anendophyte that confers increased ability to liberate and/or otherwiseprovide to the plant with nutrients selected from the group consistingof phosphate, nitrogen, potassium, iron, manganese, calcium, molybdenum,vitamins, or other micronutrients. Such a plant can exhibit increasedgrowth in soil comprising limiting amounts of such nutrients whencompared with reference agricultural plant. Differences between theendophyte-associated plant and reference agricultural plant can bemeasured by comparing the biomass of the two plant types grown underlimiting conditions, or by measuring the physical parameters describedabove. Therefore, in one embodiment, the plant comprising endophyteshows increased tolerance to nutrient limiting conditions as compared toa reference agricultural plant grown under the same nutrient limitedconcentration in the soil, as measured for example by increased biomassor seed yield of at least 10%, for example at least 15%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, atleast 100%, at least 150%, at least 200%, at least 300% or more, whencompared with uninoculated plants grown under the same conditions. Inanother embodiment, the plant containing the endophyte is able to grownunder nutrient stress conditions while exhibiting no difference in thephysiological parameter compared to a plant that is grown withoutnutrient stress. In some embodiments, such a plant will exhibit nodifference in the physiological parameter when grown with 2-5% lessnitrogen than average cultivation practices on normal agricultural land,for example, at least 5-10% less nitrogen, at least 10-15% lessnitrogen, at least 15-20% less nitrogen, at least 20-25% less nitrogen,at least 25-30% less nitrogen, at least 30-35% less nitrogen, at least35-40% less nitrogen, at least 40-45% less nitrogen, at least 45-50%less nitrogen, at least 50-55% less nitrogen, at least 55-60% lessnitrogen, at least 60-65% less nitrogen, at least 65-70% less nitrogen,at least 70-75% less nitrogen, at least 80-85% less nitrogen, at least85-90% less nitrogen, at least 90-95% less nitrogen, or less, whencompared with crop plants grown under normal conditions during anaverage growing season. In some embodiments, the microbe capable ofproviding nitrogen-stress tolerance to a plant is diazotrophic. In otherembodiments, the microbe capable of providing nitrogen-stress toleranceto a plant is non-diazotrophic.

Cold Stress.

In some cases, endophytes can confer to the plant the ability totolerate cold stress. Many known methods exist for the measurement of aplant's tolerance to cold stress (as reviewed, for example, in Thomashow(2001) Plant Physiol. 125: 89-93, and Gilmour et al. (2000) PlantPhysiol. 124: 1854-1865, both of which are incorporated herein byreference in their entirety). As used herein, cold stress refers to boththe stress induced by chilling (0° C.-15° C.) and freezing (<0° C.).Some cultivars of agricultural plants can be particularly sensitive tocold stress, but cold tolerance traits may be multigenic, making thebreeding process difficult. Endophytes able to confer cold tolerancewould potentially reduce the damage suffered by farmers on an annualbasis Barka et al. 2006, Appl. Environ. Microbiol. 72:7246-7252,incorporated herein by reference in its entirety). Improved response tocold stress can be measured by survival of plants, production ofprotectant substances such as anthocyanin, the amount of necrosis ofparts of the plant, or a change in crop yield loss, as well as thephysiological parameters used in other examples. Therefore, in oneembodiment, the plant comprising endophytes shows increased coldtolerance exhibits as compared to a reference agricultural plant grownunder the same conditions of cold stress.

Biotic Stress.

In other embodiments, the endophyte protects the plant from a bioticstress, for example, insect infestation, nematode infestation, complexinfection, fungal infection, oomycete infection, protozoal infection,viral infection, and herbivore grazing, or a combination thereof

Insect Herbivory.

There are an abundance of insect pest species that can infect or infesta wide variety of plants. Pest infestation can lead to significantdamage. Insect pests that infest plant species are particularlyproblematic in agriculture as they can cause serious damage to crops andsignificantly reduce plant yields. A wide variety of different types ofplant are susceptible to pest infestation including commercial cropssuch as cotton, soybean, wheat, barley, and corn.

In some cases, endophytes described herein may confer upon the hostplant the ability to repel insect herbivores. In other cases, endophytesmay produce, or induce the production in the plant of, compounds whichare insecticidal or insect repellant. The insect may be any one of thecommon pathogenic insects affecting plants, particularly agriculturalplants.

The endophyte-associated plant can be tested for its ability to resist,or otherwise repel, pathogenic insects by measuring, for example, insectload, overall plant biomass, biomass of the fruit or grain, percentageof intact leaves, or other physiological parameters described herein,and comparing with a reference agricultural plant. In one embodiment,the endophyte-associated plant exhibits increased biomass as compared toa reference agricultural plant grown under the same conditions (e.g.,grown side-by-side, or adjacent to, endophyte-associated plants). Inother embodiments, the endophyte-associated plant exhibits increasedfruit or grain yield as compared to a reference agricultural plant grownunder the same conditions (e.g., grown side-by-side, or adjacent to,endophyte-associated plants).

Nematodes.

Nematodes are microscopic roundworms that feed on the roots, fluids,leaves and stems of more than 2,000 row crops, vegetables, fruits, andornamental plants, causing an estimated $100 billion crop loss worldwideand accounting for 13% of global crop losses due to disease. A varietyof parasitic nematode species infect crop plants, including root-knotnematodes (RKN), cyst- and lesion-forming nematodes. Root-knotnematodes, which are characterized by causing root gall formation atfeeding sites, have a relatively broad host range and are thereforeparasitic on a large number of crop species. The cyst- andlesion-forming nematode species have a more limited host range, butstill cause considerable losses in susceptible crops.

Signs of nematode damage include stunting and yellowing of leaves, andwilting of the plants during hot periods. Nematode infestation, however,can cause significant yield losses without any obvious above-grounddisease symptoms. The primary causes of yield reduction are due tounderground root damage. Roots infected by SCN are dwarfed or stunted.Nematode infestation also can decrease the number of nitrogen-fixingnodules on the roots, and may make the roots more susceptible to attacksby other soil-borne plant nematodes.

In one embodiment, the endophyte-associated plant has an increasedresistance to a nematode when compared with a reference agriculturalplant. As before with insect herbivores, biomass of the plant or aportion of the plant, or any of the other physiological parametersmentioned elsewhere, can be compared with the reference agriculturalplant grown under the same conditions. Particularly useful measurementsinclude overall plant biomass, biomass and/or size of the fruit orgrain, and root biomass. In one embodiment, the endophyte-associatedplant exhibits increased biomass as compared to a reference agriculturalplant grown under the same conditions (e.g., grown side-by-side, oradjacent to, the endophyte-associated plants, under conditions ofnematode challenge). In another embodiment, the endophyte-associatedplant exhibits increased root biomass as compared to a referenceagricultural plant grown under the same conditions (e.g., grownside-by-side, or adjacent to, the endophyte-associated plants, underconditions of nematode challenge). In still another embodiment, theendophyte-associated plant exhibits increased fruit or grain yield ascompared to a reference agricultural plant grown under the sameconditions (e.g., grown side-by-side, or adjacent to, theendophyte-associated plants, under conditions of nematode challenge).

Fungal Pathogens.

Fungal diseases are responsible for yearly losses of over $10 Billion onagricultural crops in the US, represent 42% of global crop losses due todisease, and are caused by a large variety of biologically diversepathogens. Different strategies have traditionally been used to controlthem. Resistance traits have been bred into agriculturally importantvarieties, thus providing various levels of resistance against either anarrow range of pathogen isolates or races, or against a broader range.However, this involves the long and labor intensive process ofintroducing desirable traits into commercial lines by genetic crossesand, due to the risk of pests evolving to overcome natural plantresistance, a constant effort to breed new resistance traits intocommercial lines is required. Alternatively, fungal diseases have beencontrolled by the application of chemical fungicides. This strategyusually results in efficient control, but is also associated with thepossible development of resistant pathogens and can be associated with anegative impact on the environment. Moreover, in certain crops, such asbarley and wheat, the control of fungal pathogens by chemical fungicidesis difficult or impractical.

The present invention contemplates the use of endophytes that are ableto confer resistance to fungal pathogens to the host plant. Increasedresistance to fungal inoculation can be measured, for example, using anyof the physiological parameters presented above, by comparing withreference agricultural plants. In one embodiment, theendophyte-associated plant exhibits increased biomass and/or lesspronounced disease symptoms as compared to a reference agriculturalplant grown under the same conditions (e.g., grown side-by-side, oradjacent to, the endophyte-associated plants, infected with the fungalpathogen). In still another embodiment, the endophyte-associated plantexhibits increased fruit or grain yield as compared to a referenceagricultural plant grown under the same conditions (e.g., grownside-by-side, or adjacent to, the endophyte-associated plants, infectedwith the fungal pathogen). In another embodiment, theendophyte-associated plant exhibits decreased hyphal growth as comparedto a reference agricultural plant grown under the same conditions (e.g.,grown side-by-side, or adjacent to, the endophyte-associated plants,infected with the fungal pathogen).

Viral Pathogens.

Plant viruses are estimated to account for 18% of global crop losses dueto disease. There are numerous examples of viral pathogens affectingagricultural productivity. In one embodiment, the endophyte providesprotection against viral pathogens such that the plant has increasedbiomass as compared to a reference agricultural plant grown under thesame conditions. In still another embodiment, the endophyte-associatedplant exhibits greater fruit or grain yield, when challenged with avirus, as compared to a reference agricultural plant grown under thesame conditions. In yet another embodiment, the endophyte-associatedplant exhibits lower viral titer, when challenged with a virus, ascompared to a reference agricultural plant grown under the sameconditions.

Complex Pathogens.

Likewise, endofungal bacterial pathogens are a significant problemnegatively affecting agricultural productivity and accounting for 27% ofglobal crop losses due to plant disease. In one embodiment, theendophyte described herein provides protection against endofungalbacterial pathogens such that the plant has greater biomass as comparedto a reference agricultural plant grown under the same conditions. Instill another embodiment, the endophyte-associated plant exhibitsgreater fruit or grain yield, when challenged with a complex pathogen,as compared to a reference agricultural plant grown under the sameconditions. In yet another embodiment, the endophyte-associated plantexhibits lower complex count, when challenged with a bacterium, ascompared to a reference agricultural plant grown under the sameconditions.

Improvement of Other Traits.

In other embodiments, the endophyte can confer other beneficial traitsto the plant. Improved traits can include an improved nutritionalcontent of the plant or plant element used for human consumption. In oneembodiment, the endophyte-associated plant is able to produce adetectable change in the content of at least one nutrient. Examples ofsuch nutrients include amino acid, protein, oil (including any one ofOleic acid, Linoleic acid, Alpha-linoleic acid, Saturated fatty acids,Palmitic acid, Stearic acid and Trans fats), carbohydrate (includingsugars such as sucrose, glucose and fructose, starch, or dietary fiber),Vitamin A, Thiamine (vit. B1), Riboflavin (vit. B2), Niacin (vit. B3),Pantothenic acid (B5), Vitamin B6, Folate (vit. B9), Choline, Vitamin C,Vitamin E, Vitamin K, Calcium, Iron, Magnesium, Manganese, Phosphorus,Potassium, Sodium, Zinc. In one embodiment, the endophyte-associatedplant or part thereof contains at least one increased nutrient whencompared with reference agricultural plants.

In other cases, the improved trait can include reduced content of aharmful or undesirable substance when compared with referenceagricultural plants. Such compounds include those which are harmful wheningested in large quantities or are bitter tasting (for example, oxalicacid, amygdalin, certain alkaloids such as solanine, caffeine, nicotine,quinine and morphine, tannins, cyanide). As such, in one embodiment, theendophyte-associated plant or part thereof contains less of theundesirable substance when compared with reference agricultural plant.In a related embodiment, the improved trait can include improved tasteof the plant or a part of the plant, including the fruit or seed. In arelated embodiment, the improved trait can include reduction ofundesirable compounds produced by other endophytes in plants, such asdegradation of Fusarium-produced deoxynivalenol (also known as vomitoxinand a virulence factor involved in Fusarium head blight of maize andwheat) in a part of the plant, including the fruit or seed.

In other cases, the improved trait can be an increase in overall biomassof the plant or a part of the plant, including its fruit or seed.

The endophyte-associated plant can also have an altered hormone statusor altered levels of hormone production when compared with a referenceagricultural plant. An alteration in hormonal status may affect manyphysiological parameters, including flowering time, water efficiency,apical dominance and/or lateral shoot branching, increase in root hair,and alteration in fruit ripening.

The association between the endophyte and the plant can also be detectedusing other methods known in the art. For example, the biochemical,metabolomics, proteomic, genomic, epigenomic and/or transcriptomicprofiles of endophyte-associated plants can be compared with referenceagricultural plants under the same conditions.

Metabolomic differences between the plants can be detected using methodsknown in the art. For example, a biological sample (whole tissue,exudate, phloem sap, xylem sap, root exudate, etc.) fromendophyte-associated and reference agricultural plants can be analyzedessentially as described in Fiehn et al., (2000) Nature Biotechnol., 18,1157-1161, or Roessner et al., (2001) Plant Cell, 13, 11-29,incorporated herein by reference in its entirety. Such metabolomicmethods can be used to detect differences in levels in hormone,nutrients, secondary metabolites, root exudates, phloem sap content,xylem sap content, heavy metal content, and the like. Such methods arealso useful for detecting alterations in endophyte content and status;for example, the presence and levels of signaling molecules (e.g.,autoinducers and pheromones), which can indicate the status ofgroup-based behavior of endophytes based on, for example, populationdensity (see, for example Daniels et al., 2006, PNAS 103: 14965-14970;Eberhard et al. 1981, Biochemistry 20: 2444-2449, incorporated herein byreference in its entirety). Transcriptome analysis (reviewed, forexample, in Usadel & Fernie, 2013, Front. Plant Sci. 4:48, incorporatedherein by reference in its entirety) of endophyte-associated andreference agricultural plants can also be performed to detect changes inexpression of at least one transcript, or a set or network of genes uponendophyte association. Similarly, epigenetic changes can be detectedusing methylated DNA immunoprecipitation followed by high-throughputsequencing (Vining et al. 2013, BMC Plant Biol. 13:92, incorporatedherein by reference in its entirety).

Populations of Plant Elements

In another aspect, the invention provides for a substantially uniformpopulation of plant elements comprising a plurality of plant elementscomprising the endophytic microbial population, as described hereinabove. Substantial uniformity can be determined in many ways. In somecases, at least 10%, for example, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 75%, at least80%, at least 90%, at least 95% or more of the plant elements in thepopulation, contains the endophytic microbial population in an amounteffective to colonize the plant disposed on the surface of the plantelements. In other cases, at least 10%, for example, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 90%, at least 95% or more of the plantelements in the population, contains at least 100 CFU or spores on itssurface, for example, at least 200 CFU or spores, at least 300 CFU orspores, at least 1,000 CFU or spores, at least 3,000 CFU or spores, atleast 10,000 CFU or spores, at least 30,000 CFU or spores, at least100,000 CFU or spores, at least 300,000 CFU or spores, or at least1,000,000 CFU or spores per plant element or more.

The synthetic combinations of the present invention may be confinedwithin an object selected from the group consisting of: bottle, jar,ampule, package, vessel, bag, box, bin, envelope, carton, container,silo, shipping container, truck bed, and case. In a particularembodiment, the population of plant elements is packaged in a bag orcontainer suitable for commercial sale. For example, a bag contains aunit weight or count of the plant elements comprising the endophyticmicrobial population as described herein, and further comprises a label.In one embodiment, the bag or container contains at least 1,000 plantelements, for example, at least 5,000 plant elements, at least 10,000plant elements, at least 20,000 plant elements, at least 30,000 plantelements, at least 50,000 plant elements, at least 70,000 plantelements, at least 80,000 plant elements, at least 90,000 plant elementsor more. In another embodiment, the bag or container can comprise adiscrete weight of plant elements, for example, at least 1 lb, at least2 lbs, at least 5 lbs, at least 10 lbs, at least 30 lbs, at least 50lbs, at least 70 lbs or more. The bag or container comprises a labeldescribing the plant elements and/or said endophytic microbialpopulation. The label can contain additional information, for example,the information selected from the group consisting of: net weight, lotnumber, geographic origin of the plant elements, test date, germinationrate, inert matter content, and the amount of noxious weeds, if any.Suitable containers or packages include those traditionally used inplant element commercialization. The invention also contemplates othercontainers with more sophisticated storage capabilities (e.g., withmicrobiologically tight wrappings or with gas- or water-proofcontainments).

In some cases, a sub-population of plant elements comprising theendophytic microbial population is further selected on the basis ofincreased uniformity, for example, on the basis of uniformity ofmicrobial population. For example, individual plant elements of poolscollected from individual cobs, individual plants, individual plots(representing plants inoculated on the same day) or individual fieldscan be tested for uniformity of microbial density, and only those poolsmeeting specifications (e.g., at least 80% of tested plant elements haveminimum density, as determined by quantitative methods describedelsewhere) are combined to provide the agricultural plant elementsub-population.

The methods described herein can also comprise a validating step. Thevalidating step can entail, for example, growing some plant elementscollected from the inoculated plants into mature agricultural plants,and testing those individual plants for uniformity. Such validating stepcan be performed on individual plant elements collected from cobs,individual plants, individual plots (representing plants inoculated onthe same day) or individual fields, and tested as described above toidentify pools meeting the required specifications.

Populations of Plants, Agricultural Fields

A major focus of crop improvement efforts has been to select varietieswith traits that give, in addition to the highest return, the greatesthomogeneity and uniformity. While inbreeding can yield plants withsubstantial genetic identity, heterogeneity with respect to plantheight, flowering time, and time to seed, remain impediments toobtaining a homogeneous field of plants. The inevitable plant-to-plantvariability are caused by a multitude of factors, including unevenenvironmental conditions and management practices. Another possiblesource of variability can, in some cases, be due to the heterogeneity ofthe microbial population inhabit the plants. By providing endophyticmicrobial populations onto seeds and seedlings, the resulting plantsgenerated by germinating the seeds and seedlings have a more consistentmicrobial composition, and thus are expected to yield a more uniformpopulation of plants.

Therefore, in another aspect, the invention provides a substantiallyuniform population of plants. The population comprises at least 100plants, for example, at least 300 plants, at least 1,000 plants, atleast 3,000 plants, at least 10,000 plants, at least 30,000 plants, atleast 100,000 plants or more. The plants are grown from the seedscomprising the endophytic microbial population as described herein. Theincreased uniformity of the plants can be measured in a number ofdifferent ways.

In one embodiment, there is an increased uniformity with respect to themicrobes within the plant population. For example, in one embodiment, asubstantial portion of the population of plants, for example at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 90%, at least95% or more of the seeds or plants in a population, contains a thresholdnumber of the endophytic microbial population. The threshold number canbe at least 100 CFU or spores, for example at least 300 CFU or spores,at least 1,000 CFU or spores, at least 3,000 CFU or spores, at least10,000 CFU or spores, at least 30,000 CFU or spores, at least 100,000CFU or spores or more, in the plant or a part of the plant.Alternatively, in a substantial portion of the population of plants, forexample, in at least 1%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 90%, at least 95% or more of the plants in thepopulation, the endophytic microbial population that is provided to theseed or seedling represents at least 10%, least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or 100% of the total microbepopulation in the plant/seed.

In another embodiment, there is an increased uniformity with respect toa physiological parameter of the plants within the population. In somecases, there can be an increased uniformity in the height of the plantswhen compared with a population of reference agricultural plants grownunder the same conditions. For example, there can be a reduction in thestandard deviation in the height of the plants in the population of atleast 2%, for example at least 3%, at least 4%, at least 5%, at least6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%,at least 20%, at least 30%, at least 40%, at least 50%, at least 60% ormore, when compared with a population of reference agricultural plantsgrown under the same conditions. In other cases, there can be areduction in the standard deviation in the flowering time of the plantsin the population of at least 2%, for example at least 3%, at least 4%,at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, atleast 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60% or more, when compared with a population ofreference agricultural plants grown under the same conditions.

Commodity Plant Product

The present invention provides a commodity plant product, as well asmethods for producing a commodity plant product, that is derived from aplant of the present invention. As used herein, a “commodity plantproduct” refers to any composition or product that is comprised ofmaterial derived from a plant, seed, plant cell, or plant part of thepresent invention. Commodity plant products may be sold to consumers andcan be viable or nonviable. Nonviable commodity products include but arenot limited to nonviable seeds and grains; processed seeds, seed parts,and plant parts; dehydrated plant tissue, frozen plant tissue, andprocessed plant tissue; seeds and plant parts processed for animal feedfor terrestrial and/or aquatic animal consumption, oil, meal, flour,flakes, bran, fiber, paper, tea, coffee, silage, crushed of whole grain,and any other food for human or animal consumption; and biomasses andfuel products; and raw material in industry. Industrial uses of oilsderived from the agricultural plants described herein includeingredients for paints, plastics, fibers, detergents, cosmetics,lubricants, and biodiesel fuel. Soybean oil may be split,inter-esterified, sulfurized, epoxidized, polymerized, ethoxylated, orcleaved. Designing and producing soybean oil derivatives with improvedfunctionality and improved oliochemistry is a rapidly growing field. Thetypical mixture of triglycerides is usually split and separated intopure fatty acids, which are then combined with petroleum-derivedalcohols or acids, nitrogen, sulfonates, chlorine, or with fattyalcohols derived from fats and oils to produce the desired type of oilor fat. Commodity plant products also include industrial compounds, suchas a wide variety of resins used in the formulation of adhesives, films,plastics, paints, coatings and foams.

In some cases, commodity plant products derived from the plants, orusing the methods of the present invention can be identified readily. Insome cases, for example, the presence of viable endophytic microbes canbe detected using the methods described herein elsewhere. In othercases, particularly where there are no viable endophytic microbes, thecommodity plant product may still contain at least a detectable amountof the specific and unique DNA corresponding to the microbes describedherein. Any standard method of detection for polynucleotide moleculesmay be used, including methods of detection disclosed herein.

Throughout the specification, the word “comprise,” or variations such as“comprises” or “comprising,” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers.

Methods of Using Endophytes and Synthetic Compositions ComprisingEndophytes

As described herein, purified endophyte populations and compositionscomprising the same (e.g., formulations) can be used to conferbeneficial traits to the host plant including, for example, one or moreof the following: altered oil content, altered protein content, alteredseed carbohydrate composition, altered seed oil composition, and alteredseed protein composition, chemical tolerance, cold tolerance, delayedsenescence, disease resistance, drought tolerance, ear weight, growthimprovement, health enhancement, heat tolerance, herbicide tolerance,herbivore resistance, improved nitrogen fixation, improved nitrogenutilization, improved root architecture, improved water use efficiency,increased biomass, increased root length, increased seed weight,increased shoot length, increased yield, increased yield underwater-limited conditions, kernel mass, kernel moisture content, metaltolerance, number of ears, number of kernels per ear, number of pods,nutrition enhancement, pathogen resistance, pest resistance,photosynthetic capability improvement, salinity tolerance, stay-green,vigor improvement, increased dry weight of mature seeds, increased freshweight of mature seeds, increased number of mature seeds per plant,increased chlorophyll content, increased number of pods per plant,increased length of pods per plant, reduced number of wilted leaves perplant, reduced number of severely wilted leaves per plant, and increasednumber of non-wilted leaves per plant, a detectable modulation in thelevel of a metabolite, a detectable modulation in the level of atranscript, and a detectable modulation in the proteome relative to areference plant. For example, in some embodiments, a purified endophytepopulation can improve two or more such beneficial traits, e.g., wateruse efficiency and increased tolerance to drought. Such traits can beheritable by progeny of the agricultural plant to which endophyte wasapplied or by progeny of the agricultural plant that was grown from theseed associated with endophyte.

In one aspect of the invention, the endophytes impart to the host plantan improved ability to cope with water-limited conditions.

In some cases, the endophyte may produce one or more compounds and/orhave one or more activities, e.g., one or more of the following:production of a metabolite, production of a phytohormone such as auxin,production of acetoin, production of an antimicrobial compound,production of a siderophore, production of a cellulase, production of apectinase, production of a chitinase, production of a xylanase, nitrogenfixation, or mineral phosphate solubilization. For example, an endophytecan produce a phytohormone selected from the group consisting of anauxin, a cytokinin, a gibberellin, ethylene, a brassinosteroid, andabscisic acid. In one particular embodiment, the endophyte producesauxin (e.g., indole-3-acetic acid (IAA)). Production of auxin can beassayed as described herein. Many of the microbes described herein arecapable of producing the plant hormone auxin indole-3-acetic acid (IAA)when grown in culture. Auxin plays a key role in altering the physiologyof the plant, including the extent of root growth. Therefore, in anotherembodiment, the endofungal endophytic population is disposed on thesurface or within a tissue of the seed or seedling in an amounteffective to detectably increase production of auxin in the agriculturalplant when compared with a reference agricultural plant. In oneembodiment, the increased auxin production can be detected in a tissuetype selected from the group consisting of the root, shoot, leaves, andflowers.

In some embodiments, the endophyte can produce a compound withantimicrobial properties. For example, the compound can haveantibacterial properties, as determined by the growth assays providedherein. In one embodiment, the compound with antibacterial propertiesshows bacteriostatic or bactericidal activity against E. coli and/orBacillus sp. In another embodiment, the endophyte produces a compoundwith antifungal properties, for example, fungicidal or fungistaticactivity against S. cerevisiae and/or Rhizoctonia.

In some embodiments, the endophyte is a fungus capable of nitrogenfixation, and is thus capable of producing ammonium from atmosphericnitrogen. The ability of a fungus to fix nitrogen can be confirmed bytesting for growth of the fungus in nitrogen-free growth media, forexample, LGI media, as described herein.

In some embodiments, the endophyte can produce a compound that increasesthe solubility of mineral phosphate in the medium, i.e., mineralphosphate solubilization, for example, using the growth assays describedherein. In one embodiment, the endophyte produces a compound that allowsthe bacterium to grow in growth media comprising Ca₃HPO₄ as the solephosphate source.

In some embodiments, the endophyte can produce a siderophore.Siderophores are small high-affinity iron chelating agents secreted bymicroorganisms that increase the bioavailability of iron. Siderophoreproduction by the endophyte can be detected, for example, using themethods described herein, as well as elsewhere (Perez-Miranda et al.,2007, J Microbiol Methods. 70:127-31, incorporated herein by referencein its entirety).

In some embodiments, the endophyte can produce a hydrolytic enzyme. Forexample, in one embodiment, an endophyte can produce a hydrolytic enzymeselected from the group consisting of a cellulase, a pectinase, achitinase and a xylanase. Hydrolytic enzymes can be detectedusing themethods described herein (see also, cellulase: Quadt-Hallmann et al.,(1997) Can. J. Microbiol., 43: 577-582; pectinase: Soares et al. (1999).Revista de Microbiolgia 30(4): 299-303; chitinase: Li et al., (2004)Mycologia 96: 526-536; and xylanase: Suto et al., (2002) J BiosciBioeng. 93:88-90, each of which is incorporated by reference in itsentirety).

In some embodiment, synthetic combinations comprise synergisticendofungal endophytic populations. As used herein, the term “synergisticendophytic populations” refers to two or more endophyte populations thatproduce one or more effects (e.g., two or more or three or more effects)that are greater than the sum of their individual effects. For example,in some embodiments, a purified endophyte population contains two ormore different endophytes that are capable of synergistically increasingat least one of e.g., production of a phytohormone such as auxin,production of acetoin, production of an antimicrobial compound,production of a siderophore, production of a cellulase, production of apectinase, production of a chitinase, production of a xylanase, nitrogenfixation, or mineral phosphate solubilization in an agricultural plant.Synergistically increasing one or more of such properties can increase abeneficial trait in an agricultural plant, such as an increase indrought tolerance.

In some embodiments, a purified endofungal population comprising one ormore endophytes can increase one or more properties such as productionof a phytohormone such as auxin, production of acetoin, production of anantimicrobial compound, production of a siderophore, production of acellulase, production of a pectinase, production of a chitinase,production of a xylanase, or mineral phosphate solubilization in anagricultural plant, without increasing nitrogen fixation in theagricultural plant.

In some embodiments, metabolites in plants can be modulated by makingsynthetic combinations of purified endophytic populations. For example,an endophyte described herein can cause a detectable modulation (e.g.,an increase or decrease) in the level of various metabolites, e.g.,indole-3-carboxylic acid, trans-zeatin, abscisic acid, phaseic acid,indole-3-acetic acid, indole-3-butyric acid, indole-3-acrylic acid,jasmonic acid, jasmonic acid methyl ester, dihydrophaseic acid,gibberellin A3, salicylic acid, upon colonization of a plant.

In some embodiments, the endophyte modulates the level of the metabolitedirectly (e.g., the microbe itself produces the metabolite, resulting inan overall increase in the level of the metabolite found in the plant).In other cases, the agricultural plant, as a result of the associationwith the endophytic microbe (e.g., an endophyte), exhibits a modulatedlevel of the metabolite (e.g., the plant reduces the expression of abiosynthetic enzyme responsible for production of the metabolite as aresult of the microbe inoculation). In still other cases, the modulationin the level of the metabolite is a consequence of the activity of boththe microbe and the plant (e.g., the plant produces increased amounts ofthe metabolite when compared with a reference agricultural plant, andthe endophytic microbe also produces the metabolite). Therefore, as usedherein, a modulation in the level of a metabolite can be an alterationin the metabolite level through the actions of the microbe and/or theinoculated plant.

The levels of a metabolite can be measured in an agricultural plant, andcompared with the levels of the metabolite in a reference agriculturalplant, and grown under the same conditions as the inoculated plant. Theuninoculated plant that is used as a reference agricultural plant is aplant that has not been applied with a formulation with the endophyticmicrobe (e.g., a formulation comprising a population of purifiedendophytes). The uninoculated plant used as the reference agriculturalplant is generally the same species and cultivar as, and is isogenic to,the inoculated plant.

The metabolite whose levels are modulated (e.g., increased or decreased)in the endophyte-associated plant may serve as a primary nutrient (i.e.,it provides nutrition for the humans and/or animals who consume theplant, plant tissue, or the commodity plant product derived therefrom,including, but not limited to, a sugar, a starch, a carbohydrate, aprotein, an oil, a fatty acid, or a vitamin). The metabolite can be acompound that is important for plant growth, development or homeostasis(for example, a phytohormone such as an auxin, cytokinin, gibberellin, abrassinosteroid, ethylene, or abscisic acid, a signaling molecule, or anantioxidant). In other embodiments, the metabolite can have otherfunctions. For example, in one embodiment, a metabolite can havebacteriostatic, bactericidal, fungistatic, fungicidal or antiviralproperties. In other embodiments, the metabolite can haveinsect-repelling, insecticidal, nematode-repelling, or nematicidalproperties. In still other embodiments, the metabolite can serve a rolein protecting the plant from stresses, may help improve plant vigor orthe general health of the plant. In yet another embodiment, themetabolite can be a useful compound for industrial production. Forexample, the metabolite may itself be a useful compound that isextracted for industrial use, or serve as an intermediate for thesynthesis of other compounds used in industry. In a particularembodiment, the level of the metabolite is increased within theagricultural plant or a portion thereof such that it is present at aconcentration of at least 0.1 ug/g dry weight, for example, at least 0.3ug/g dry weight, 1.0 ug/g dry weight, 3.0 ug/g dry weight, 10 ug/g dryweight, 30 ug/g dry weight, 100 ug/g dry weight, 300 ug/g dry weight, 1mg/g dry weight, 3 mg/g dry weight, 10 mg/g dry weight, 30 mg/g dryweight, 100 mg/g dry weight or more, of the plant or portion thereof.

Likewise, the modulation can be a decrease in the level of a metabolite.The reduction can be in a metabolite affecting the taste of a plant or acommodity plant product derived from a plant (for example, a bittertasting compound), or in a metabolite which makes a plant or theresulting commodity plant product otherwise less valuable (for example,reduction of oxalate content in certain plants, or compounds which aredeleterious to human and/or animal health). The metabolite whose levelis to be reduced can be a compound that affects quality of a commodityplant product (e.g., reduction of lignin levels).

In some embodiments, the endophyte is capable of generating a complexnetwork in the plant or surrounding environment of the plant, whichnetwork is capable of causing a detectable modulation in the level of ametabolite in the host plant.

In a particular embodiment, the metabolite can serve as a signaling orregulatory molecule. The signaling pathway can be associated with aresponse to a stress, for example, one of the stress conditions selectedfrom the group consisting of drought stress, salt stress, heat stress,cold stress, low nutrient stress, nematode stress, insect herbivorystress, fungal pathogen stress, complex pathogen stress, and viralpathogen stress.

The inoculated agricultural plant is grown under conditions such thatthe level of one or more metabolites is modulated in the plant, whereinthe modulation is indicative of increased resistance to a stressselected from the group consisting of drought stress, salt stress, heatstress, cold stress, low nutrient stress, nematode stress, insectherbivory stress, fungal pathogen stress, complex pathogen stress, andviral pathogen stress. The increased resistance can be measured at about10 minutes after applying the stress, for example about 20 minutes, 30minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours,about 8 hours, about 12 hours, about 16 hours, about 20 hours, about 24hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours,about 120 hours, or about a week after applying the stress.

The metabolites or other compounds described herein can be detectedusing any suitable method including, but not limited to gelelectrophoresis, liquid and gas phase chromatography, either alone orcoupled to mass spectrometry (See, for example, the Examples sectionsbelow), NMR (See e.g., U.S. patent publication 20070055456, which isincorporated herein by reference in its entirety), immunoassays(enzyme-linked immunosorbent assays (ELISA)), chemical assays,spectroscopy and the like. In some embodiments, commercial systems forchromatography and NMR analysis are utilized.

In other embodiments, metabolites or other compounds are detected usingoptical imaging techniques such as magnetic resonance spectroscopy(MRS), magnetic resonance imaging (MRI), CAT scans, ultra sound,MS-based tissue imaging or X-ray detection methods (e.g., energydispersive x-ray fluorescence detection).

Any suitable method may be used to analyze the biological sample (e.g.,seed or plant tissue) in order to determine the presence, absence orlevel(s) of the one or more metabolites or other compounds in thesample. Suitable methods include chromatography (e.g., HPLC, gaschromatography, liquid chromatography), mass spectrometry (e.g., MS,MS-MS), LC-MS, enzyme-linked immunosorbent assay (ELISA), antibodylinkage, other immunochemical techniques, biochemical or enzymaticreactions or assays, and combinations thereof. The levels of one or moreof the recited metabolites or compounds may be determined in the methodsof the present invention. For example, the level(s) of one metabolitesor compounds, two or more metabolites, three or more metabolites, fouror more metabolites, five or more metabolites, six or more metabolites,seven or more metabolites, eight or more metabolites, nine or moremetabolites, ten or more metabolites, or compounds etc., including acombination of some or all of the metabolites or compounds including,but not limited to those disclosed herein may be determined and used insuch methods.

As shown in the Examples and otherwise herein, endophyte-inoculatedplants display altered oil content, altered protein content, alteredseed carbohydrate composition, altered seed oil composition, and alteredseed protein composition, chemical tolerance, cold tolerance, delayedsenescence, disease resistance, drought tolerance, ear weight, growthimprovement, health enhancement, heat tolerance, herbicide tolerance,herbivore resistance, improved nitrogen fixation, improved nitrogenutilization, improved root architecture, improved water use efficiency,increased biomass, increased root length, increased seed weight,increased shoot length, increased yield, increased yield underwater-limited conditions, kernel mass, kernel moisture content, metaltolerance, number of ears, number of kernels per ear, number of pods,nutrition enhancement, pathogen resistance, pest resistance,photosynthetic capability improvement, salinity tolerance, stay-green,vigor improvement, increased dry weight of mature seeds, increased freshweight of mature seeds, increased number of mature seeds per plant,increased chlorophyll content, increased number of pods per plant,increased length of pods per plant, reduced number of wilted leaves perplant, reduced number of severely wilted leaves per plant, and increasednumber of non-wilted leaves per plant, a detectable modulation in thelevel of a metabolite, a detectable modulation in the level of atranscript, and a detectable modulation in the proteome relative to areference plant, or a combination thereof. Therefore, in one embodiment,the endofungal endophytic population is disposed on the surface or on orwithin a tissue of the seed or seedling in an amount effective toincrease the biomass of the plant, or a part or tissue of the plantgrown from the seed or seedling. The increased biomass is useful in theproduction of commodity products derived from the plant. Such commodityproducts include an animal feed, a fish fodder, a cereal product, aprocessed human-food product, a sugar or an alcohol. Such products maybe a fermentation product or a fermentable product, one such exemplaryproduct is a biofuel. The increase in biomass can occur in a part of theplant (e.g., the root tissue, shoots, leaves, etc.), or can be anincrease in overall biomass when compared with a reference agriculturalplant. Such increase in overall biomass can be under relativelystress-free conditions. In other cases, the increase in biomass can bein plants grown under any number of abiotic or biotic stresses,including drought stress, salt stress, heat stress, cold stress, lownutrient stress, nematode stress, insect herbivory stress, fungalpathogen stress, complex pathogen stress, and viral pathogen stress.

In another embodiment, the endofungal endophytic population is disposedon the surface or within a tissue of the seed or seedling in an amounteffective to increase the rate of seed germination when compared with areference agricultural plant.

In other cases, the endofungal microbe is disposed on the seed orseedling in an amount effective to increase the average biomass of thefruit or cob from the resulting plant when compared with a referenceagricultural plant.

Plants inoculated with an endofungal endophytic population may also showan increase in overall plant height. Therefore, in one embodiment, thepresent invention provides for a seed comprising an endofungalendophytic population that is disposed on the surface or within a tissueof the seed or seedling in an amount effective to increase the height ofthe plant. For example, the endofungal endophytic population is disposedin an amount effective to result in an increase in height of theagricultural plant when compared with a reference agricultural plant.Such an increase in height can be under relatively stress-freeconditions. In other cases, the increase in height can be in plantsgrown under any number of abiotic or biotic stresses, including droughtstress, salt stress, heat stress, cold stress, low nutrient stress,nematode stress, insect herbivory stress, fungal pathogen stress,complex pathogen stress, or viral pathogen stress.

The host plants inoculated with the endofungal endophytic population mayalso show dramatic improvements in their ability to utilize water moreefficiently. Water use efficiency is a parameter often correlated withdrought tolerance. Water use efficiency (WUE) is a parameter oftencorrelated with drought tolerance, and is the CO₂ assimilation rate peramount of water transpired by the plant. An increase in biomass at lowwater availability may be due to relatively improved efficiency ofgrowth or reduced water consumption. In selecting traits for improvingcrops, a decrease in water use, without a change in growth would haveparticular merit in an irrigated agricultural system where the waterinput costs were high. An increase in growth without a correspondingjump in water use would have applicability to all agricultural systems.In many agricultural systems where water supply is not limiting, anincrease in growth, even if it came at the expense of an increase inwater use also increases yield.

When soil water is depleted or if water is not available during periodsof drought, crop yields are restricted. Plant water deficit develops iftranspiration from leaves exceeds the supply of water from the roots.The available water supply is related to the amount of water held in thesoil and the ability of the plant to reach that water with its rootsystem. Transpiration of water from leaves is linked to the fixation ofcarbon dioxide by photosynthesis through the stomata. The two processesare positively correlated so that high carbon dioxide influx throughphotosynthesis is closely linked to water loss by transpiration. Aswater transpires from the leaf, leaf water potential is reduced and thestomata tend to close in a hydraulic process limiting the amount ofphotosynthesis. Since crop yield is dependent on the fixation of carbondioxide in photosynthesis, water uptake and transpiration arecontributing factors to crop yield. Plants which are able to use lesswater to fix the same amount of carbon dioxide or which are able tofunction normally at a low water potential, are more efficient andthereby are able to produce more biomass and economic yield in manyagricultural systems. An increased water use efficiency of the plantrelates in some cases to an increased fruit/kernel size or number.

Therefore, in one embodiment, the plants described herein exhibit anincreased water use efficiency (WUE) when compared with a referenceagricultural plant grown under the same conditions. Such an increase inWUE can occur under conditions without water deficit, or underconditions of water deficit, for example, when the soil water content isless than or equal to 60% of water saturated soil, for example, lessthan or equal to 50%, less than or equal to 40%, less than or equal to30%, less than or equal to 20%, less than or equal to 10% of watersaturated soil on a weight basis. In some embodiments, the plantsinoculated with the endofungal endophytic population show increasedyield under non-irrigated conditions, as compared to referenceagricultural plants grown under the same conditions.

In a related embodiment, the plant comprising endophyte can have ahigher relative water content (RWC), than a reference agricultural plantgrown under the same conditions.

Although the present invention has been described in detail withreference to examples below, it is understood that various modificationscan be made without departing from the spirit of the invention. Forinstance, while the particular examples below may illustrate the methodsand embodiments described herein using a specific plant, the principlesin these examples may be applied to any agricultural crop. Therefore, itwill be appreciated that the scope of this invention is encompassed bythe embodiments of the inventions recited herein and the specificationrather than the specific examples that are exemplified below. All citedpatents and publications referred to in this application are hereinincorporated by reference in their entirety.

EXAMPLES Example 1 Cultivation-Independent Analysis of Microbial Taxa inAgriculturally Relevant Seed Communities Based on Marker GeneHigh-Throughput Sequencing Example Description

Microbial taxa found in agriculturally relevant communities wereidentified using high-throughput marker gene sequencing across severalcrops and numerous varieties of seeds.

Experimental Description

We employed high-throughput sequencing of marker genes for bacteria,archaea, and fungi on seeds of 50 commercial, 22 wild, and 33 landracecultivars of corn; 40 commercial, 13 wild, and 23 landrace cultivars ofwheat; 13 cotton seeds; and 24 soybean seeds. Non-commercial varietieswere obtained from USDA GRIN through their National Plant Germplasmsystem (http://www.ars-grin.gov/npgs/). Accessions were categorized intolandrace, wild, and inbred varieties based on the assessment ofimprovement status. In order to extract microbial DNA, the seeds werefirst soaked in sterile, DNA-free water for 48 h to soften them, andthey were surface sterilized using 95% ethanol to reduce superficialcontaminant microbes. The seeds were then ground using a mortar andpestle treated with 95% ethanol and RNAse Away (Life Technologies, Inc.,Grand Island, N.Y.) to remove contaminant DNA. DNA was extracted fromthe ground seeds using the PowerPlant Pro DNA extraction kit (Mo BioLaboratories, Inc., Carlsbad, Calif.) according to the manufacturer'sinstructions.

Marker genes were amplified and sequenced from the extracted DNA using ahigh-throughput protocol similar to (Fierer et al. 2012, McGuire et al.2013). For the bacterial and archaeal analyses, the V4 hypervariableregion of the 16S rRNA gene was targeted (primers 515f/806r), and forfungi, the first internal transcribed spacer (ITS1) region of the rRNAoperon (primers ITS1f/ITS2r) was targeted. The two marker genes were PCRamplified separately using 35 cycles, and error-correcting 12-bpbarcoded primers specific to each sample were used to facilitatecombining of samples. To reduce the amplification of chloroplast andmitochondrial DNA, we used PNA clamps specific to the rRNA genes inthese organelles (Lundberg et al. 2013). PCR reactions to amplify 16SrRNA genes followed the protocol of (Lundberg et al. 2013), and those toamplify ITS regions followed the protocol of (Fierer et al. 2012). PCRproducts were quantified using the PicoGreen assay (Life Technologies,Inc., Grand Island, N.Y.), pooled in equimolar concentrations, andcleaned using the UltraClean kit (Mo Bio Laboratories, Inc., Carlsbad,Calif.). Cleaned DNA pools were sequenced on an Illumina MiSeqinstrument at the University of Colorado Next Generation SequencingFacility.

Old OTU Assignment

The raw sequence data were reassigned to distinct samples using a customPython script, and quality filtering and OTU (operational taxonomicunit) clustering was conducted using the UPARSE pipeline (Edgar 2013).Briefly, a de novo sequence database with representative sequences foreach OTU was created using a 97% similarity threshold, and raw readswere mapped to this database to calculate sequence counts per OTU persample. Prior to creating the database, sequences were quality filteredusing an expected error frequency threshold of 0.5 errors per sequence.In addition, sequences were dereplicated and singletons were removedprior to creating the database. OTUs were provided taxonomicclassifications using the RDP classifier (Wang et al. 2007) trained withthe Greengenes (McDonald et al. 2012) or UNITE (Abarenkov et al. 2010)databases for 16S rRNA and ITS sequences, respectively. To account fordifferences in the number of sequences per sample, each sample wasrarefied to 1,000 and 6,500 sequences per sample for 16S rRNA and ITSdatasets. This resulted in samples with fewer sequences than therarefaction depth to be discarded from downstream analyses. OTUsclassified as chloroplasts or mitochondria were discarded prior torarefaction.

New OTU Assignment

For both 16S rRNA and ITS1 sequences, we used barcoded primers unique toeach sample to combine multiple samples in an Illumina MiSeq run. Theresulting reads were separated back into their respective samples basedon the barcodes using a custom Python script. We performed qualityfiltering following the UPARSE pipeline (Edgar, 2013), including mergingpaired end reads, setting a maximum expected error rate of <=1 error permerged sequence, and removing singletons (reads occurring only oneacross all samples in a run).

The original de novo OTU (operational taxonomic units) clustering wasperformed at 97% sequence similarity, again following the UPARSEpipeline. Subsequent New OTU (new OTU) clustering (Rideout et al, 2014)was performed using a cascading approach, comparing the sequencesagainst the Greengenes (McDonald et al, 2012) and UNITE (Abardenkov etal, 2010) databases, which are provided with full-length clustering atvarious widths. Bacterial sequences were first compared to theGreengenes 99% OTU representative sequences. Sequences without a 99%match to the 99% OTUs were then compared to the Greengenes 97% OTUs at97%. Fungal sequences were first compared to the UNITE Dynamic OTUrepresentative sequences, where dynamic represents values between 97%and 99% depending on the OTU. Sequences that did not match the UNITEDynamic OTUs at the appropriate clustering level, were compared to theUNITE 97% OTUs at 97%. The remaining sequences that did not match eitherGreengenes or UNITE, and were present at a level of at least 10 readsacross the samples, were clustered using the de novo method above(independently for the bacterial and fungal sequences). The originalsequences were mapped to the New OTUs using the same cascading approach,and any sequences that did not match an OTU, but did match a sequencewith fewer than 10 copies were designated with the read ID representingthat unique sequence.

The original de novo OTUs were provided taxonomic classifications usingthe RDP classifier (Wang et al. 2007) trained with the Greengenes(McDonald et al. 2012) and UNITE (Abarenkov et al. 2010) databases for16S rRNA and ITS sequences, respectively. To account for differences inthe variable number of sequences per sample, each sample was rarefied to1000 16S rRNA and 1000 ITS sequences per sample. OTUs classified aschloroplasts or mitochondria were discarded prior to rarefaction.

Overall differences in bacterial community composition between thecontrol and inoculated plants were evaluated using non-metricmultidimensional scaling based on Bray-Curtis dissimilarities in orderto visualize pairwise differences between sample communities.Permutational analysis of variance (PERMANOVA) was used to statisticallytest the significance of these differences. Analyses were conductedusing the vegan package in R (R Core Team 2013). To determine the OTUscontributing to overall differences among crop types, mean relativeabundances were calculated for each OTU within each crop type. Only OTUswith a mean relative abundance of 0.1% in either group were included inthis analysis. The tables demonstrating presence absence wereconstructed using the New OTUs, assessing the presence of each OTU inany of the sample replicates, and reporting only the OTUs matching therelevant sequences.

Example Results: Core Taxa

OTUs were determined to be core taxa based on detection across a varietyof seed types. For example, taxa core across crops were those present inseeds from >2 crops. Similarly, taxa core to an individual crop werethose present in seeds from >2 cultivar categories (i.e. wild, landrace,inbred, or modern) within that crop. In an effort to conservativelyselect extant core taxa, OTUs where at least class level taxonomy couldbe resolved were discarded. Representative strains from our straincollection for each OTU were determined when possible using 16S rRNAgene clustering at the 97% similarity threshold in USEARCH (Edgar 2010).

Across seeds from all crops (corn, wheat, cotton, and soybean), 2,697bacterial and 415 fungal OTUs were detected and evaluated following ourstringent sequence quality filtering approach. Fungal sequences were notdetectable in soybean samples, and thus, analyses across fungal taxawere conducted within the 3 remaining crops.

Within cotton, 176 bacterial and 68 fungal OTUs were found (Table 1B).Among these, 50 taxa, consisting of 25 bacterial and 25 fungal OTUs,were found only in cotton seeds, and not in seeds of corn, wheat, orsoybean (Table 1A).

Within corn, 2351 OTUs were found, including 2169 bacterial OTUs and 182fungal OTUs (Table 2C). Among these, 1964 OTUs, including 1853 bacterialOTUs and 111 fungal OTUs, were found only in corn, and not in seeds ofwheat, soybean, or cotton seeds tested (Table 2A). OTUs that were foundto co-occur within corn seeds with a correlation of more than 0.5 arefound in Table 2B.

Within soybeans, 1097 bacterial OTUs were found (Table 3C). Among these,367 bacterial taxa were found only in soybean, and not in seeds of corn,wheat, or cotton seeds tested (Table 3A). OTUs that were found toco-occur within soy seeds with a correlation of more than 0.5 are foundin Table 3B.

Within wheat, 557 OTUs were found, including 354 bacterial OTUs and 203fungal OTUs (Table 4C). Among these, 226 OTUs, including 85 bacterialOTUs and 149 fungal OTUs, were found only in wheat, and not in seeds ofcorn, soybean, or cotton seeds tested (Table 4A). OTUs that were foundto co-occur within wheat seeds with a correlation of more than 0.5 arefound in Table 4B.

Example Results: Ancestral Vs. Modern Taxa

Overall bacterial and fungal community compositions were comparedbetween ancestral and modern seeds by first visualizing differencesusing non-metric multidimensional scaling based on Bray-Curtisdissimilarities. Statistical significance of differences was testedusing permutational multivariate analysis of variance (PERMANOVA) withthe vegan package in R (R Core Team 2013 R: A Language and Environmentfor Statistical Computing. R Foundation for Statistical Computing,Vienna, Austria ISBN: 3-900051-07-0. Available online athttp://www.R-project.org/). The Shannon-Wiener diversity index was alsocalculated with the vegan package in R. OTUs having greater associationswith ancestral seed types compared to modern seeds were identified usingcomparisons of the relative abundances of OTUs. Specifically, sequencecounts per OTU were converted to relative abundances and median relativeabundances were calculated for each seed type. We assessed differencesbetween ancestral and modern seeds when median relative abundanceswere >0.1%. OTUs without taxonomy resolved to at least class level wereremoved from analysis. Representative strains from the current straincollection were found fNew OTUs when possible using 16S rRNA sequenceclustering at the 97% threshold in USEARCH v7.0 [Edgar (2010) Naturemethods 10:996-8, incorporated herein by reference]. Bacterial communitycomposition significantly differed between wild and modern seeds forboth corn (P<0.001; FIG. 1A) and wheat (P<0.001; FIG. 1B). This was alsothe case when landrace and modern seeds were compared (P<0.05).

Among corn seeds, 14 bacterial OTUs were overrepresented in wildcompared to modern seed varieties. These taxa included several membersof the Enterobacteriaceae family as well as Paenibacillaceae,Planococcaceae, and Oxalobacteraceae (Table 16). Similarly, six OTUswere overrepresented in landrace compared to modern corn seeds. Thesealso included several Enterobacteriaceae as well as one Xanthomonadaceae(Table 17). All these differences in composition were translated intodifferences in diversity, with modern corn being significantly morediverse than Teosinte and the Landrace (FIG. 3).

Among wheat seeds, 5 bacterial OTUs were overrepresented in wildcompared to modern seed varieties (Table 18). 3 OTUs wereoverrepresented in landrace compared to modern wheat seeds. There taxawere members of the Enterobacteriaceae (OTU_(—)3078, OTU_(—)2, andOTU_(—)2912). (Table 19). The diversity of the bacterial community washigher in modern wheat than in the Landrace (FIG. 4).

Seed fungal communities were also diverse, although less so thanbacterial communities (FIGS. 5 and 6). The pattern of diversity wasopposite to that observed among the bacterial communities, with themodern varieties containing the least diverse fungal communities. Over500 unique OTUs were observed across all samples. Fungal communitycomposition significantly differed between wild and modern seeds fromwheat P<0.001; Fig. XB). This was also the case for landrace and modernseeds from wheat. Differences between wild and modern seeds were notsignificantly different for corn (P>0.01; Fig. XA), but this was likelydue to insufficient replication.

Among corn seeds, 1 fungal OTU was overrepresented in wild compared tomodern seed varieties. This OTU was classified as an Acremonium species(Table 20). 2 fungal OTUs were overrepresented in landrace compared tomodern seed varieties. Both OTUs were members of the Sordariomycetes andone was the same Acremonium species as that overrepresented in wildcompared to modern seeds (Table 21).

Among wheat seeds, 7 fungal OTUs were overrepresented in wild comparedto modern seed varieties. These OTUs were all members of theDothideomycetes and included 3 taxa identified as Cladosporium (Table22). Two of these OTUs were also overrepresented in landrace compared tomodern seed varieties (Table 23).

FIGS. 3 and 4 show the Shannon Diversity indices of bacterialcommunities found in Wild, Landrace, and modern cultivars of corn andwheat, respectively. FIGS. 5 and 6 show the Shannon Diversity indices offungal communities found in Teosinte, Landrace, Inbred, and moderncultivars of corn and wheat, respectively. Modern corn and wheat eachhad a lower diversity of fungal communities.

In conclusion, we have identified a number of bacterial and fungalmicrobes present in ancestral and landrace cultivars of wheat and cornthat are underrepresented in modern cultivars. Our analysis elucidatedseveral bacterial and fungal OTUs that were overrepresented in ancestralseeds compared to modern seeds.

Example 2 Identification of Root Endophytes Belonging to OTUs Identifiedin Seeds

In the above examples, microbial taxa core to agriculturally relevantseeds were identified. In this example, seeds from several crops andnumerous varieties were grown in the greenhouse or the field, andcommunity sequencing was performed on root samples to identify taxacorresponding to core seed taxa.

Experimental Description

The crops used in this experiment were designated: Modern Maize 1,Modern Maize 2, Landrace Maize, Wild Maize, Modern Wheat 1, Modern Wheat2, Landrace Wheat, Wild Wheat, Modern Soy 1, Modern Soy 2, Wild Soy 1,Wild Soy 2, Modern Cotton 1, Modern Cotton 2, Landrace Cotton, and WildCotton.

The purpose of this work was to use Illumina sequencing to define thebacterial and fungal endophyte populations residing in the roots ofwild, landrace, and modern varieties of maize, wheat, soybean, andcotton when grown in two different greenhouses (designated as“Massachusetts” or “Texas”) or in three geographically different fieldlocations (Minnesota and Idaho). As a negative control to promoteenrichment for seed transmitted endophytes, plants were grown inautoclaved (sterile) sand under controlled conditions in theMassachusetts greenhouse. To see if seed transmitted endophytes mightpersist in roots grown in soil, all the above listed genotypes of plantwere grown in the Massachusetts greenhouse planted with commercialnursery soil from Massachusetts. To see if some of these modernvarieties would maintain seed transmitted endophytes in roots when grownin a different greenhouse, Modern Cotton 1 and 2, Modern Soy 1 and 2,Modern Maize 1 and 2, and Modern Wheat 1 and 2 were planted in cleancontainers filled with wheat field soil from Texas, in a greenhouse inTexas. To see if some of these modern varieties would maintain seedtransmitted endophytes in roots when grown in geographically differentfield locations, Modern Cotton 1 and 2, Modern Soy 1 and 2, Modern Maize1 and 2, and Modern Wheat 1 and 2 were planted in wheat fields inMinnesota and Idaho.

New, plastic containers were filled with heat sterilized quartz sandprior to each seed being per accession being planted. For soiltreatments in Massachusetts, heat sterilized quartz sand was mixed withsoil in a ratio of 3:1. For soil treatments in Texas, containers werefilled with unmixed wheat field soil. To pre-germinate seedlings inMassachusetts, unsterilized seeds of each accession were placed onsterile sand or pure soil in a Petri dish, then watered with sterilewater. One seedling of each seedling of each accession was planted ineach of 4 sterile sand filled containers, 4 sand:soil filled containersor 4 soil filled containers. In the Texas greenhouse, seeds were placeddirectly into the soil in cups, without pre-germination. Likewise at theMinnesota and Idaho field locations, seeds were placed into the groundwithout pregermination. Greenhouse grown seedlings were watered with 25mL of sterile water every two days, while field grown plants were onlywatered once with tap water right after planting. Plants were grown for21 days in Massachusetts and then harvested, while in Texas, Minnesotaand Idaho they were grown for 14 days then harvested.

Harvesting involved shaking plants free of as much soil/debris aspossible, cutting plants into shoot and root, placing them into 15 mLconical tubes along with 10 mL of distilled water, shaken vigourously,then decanting off the dirty water. This washing step was repeated withsterile water until wash water was no longer cloudy (the last rinsecoming off of every root was clear). The washed root material in 15 mLconical tube then had added to it two sterile carbide beads and 5 mL ofsterile water before homogenizing in the Fastprep24 machine for 1 minuteat 6M vibrations per second.

DNA was extracted from this material using a PowerPlant® Pro-htp 96 DNAextraction kit (Mo Bio Laboratories, Inc., Carlsbad, Calif.) accordingto the manufacturer's instructions. Microbial composition was assessedin each sample using the methods described in Example 1.

The original de novo OTU (operational taxonomic units) clustering wasperformed at 97% sequence similarity, again following the UPARSEpipeline. Subsequent “New OTU” clustering (Rideout et al, 2014) wasperformed using a cascading approach, comparing the sequences againstthe Greengenes (McDonald et al, 2012) and UNITE (Abardenkov et al, 2010)databases, which are provided with full-length clustering at variouswidths. Bacterial sequences were first compared to the Greengenes 99%OTU representative sequences. Sequences without a 99% match to the 99%OTUs were then compared to the Greengenes 97% OTUs at 97%. Fungalsequences were first compared to the UNITE Dynamic OTU representativesequences, where dynamic represents values between 97% and 99% dependingon the OTU. Sequences that did not match the UNITE Dynamic OTUs at theappropriate clustering level, were compared to the UNITE 97% OTUs at97%. The remaining sequences that did not match either Greengenes orUNITE, and were present at a level of at least 10 reads across thesamples, were clustered using the de novo method above (independentlyfor the bacterial and fungal sequences). The original sequences weremapped to the New OTUs using the same cascading approach, and anysequences that did not match an OTU, but did match a sequence with fewerthan 10 copies were designated with the read ID representing that uniquesequence.

The original de novo OTUs were provided taxonomic classifications usingthe RDP classifier (Wang et al. 2007) trained with the Greengenes(McDonald et al. 2012) and UNITE (Abarenkov et al. 2010) databases for16S rRNA and ITS sequences, respectively. To account for differences inthe variable number of sequences per sample, each sample was rarefied to1000 16S rRNA and 1000 ITS sequences per sample. OTUs classified aschloroplasts or mitochondria were discarded prior to rarefaction.

Overall differences in bacterial community composition between thecontrol and inoculated plants were evaluated using non-metricmultidimensional scaling based on Bray-Curtis dissimilarities in orderto visualize pairwise differences between sample communities.Permutational analysis of variance (PERMANOVA) was used to statisticallytest the significance of these differences. Analyses were conductedusing the vegan package in R (R Core Team 2013). To determine the OTUscontributing to overall differences among crop types, mean relativeabundances were calculated for each OTU within each crop type. Only OTUswith a mean relative abundance of 0.1% in either group were included inthis analysis. The tables demonstrating presence absence wereconstructed using the new OTUs, assessing the presence of each OTU inany of the sample replicates, and reporting only the OTUs matching therelevant sequences.

Example Results

Experiments previously detected 2,697 bacterial and 415 fungal OTUs inseeds of corn, wheat, cotton, and soybean. Bacterial taxa represented218 families and 334 genera. Fungal taxa represented 48 families and 87genera highlighting the broad diversity of endophytic microbes withinseeds. Searching for these same OTUs in washed roots of corn, wheat,cotton, and soybeans grown in either Massachusetts or Texas greenhouseand Idaho or Minnesota field, 624 (23%) of the bacterial and 48 (12%) ofthe fungal OTUs were observed in seeds. Searching for these only inroots of plants grown in the Massachusetts greenhouse (Tables 25, 26),176 (7%) of these bacterial and 33 (8%) of these fungal OTUs weredetected. In roots of plants grown in the Texas greenhouse (Tables 27,28), 395 (15%) of these bacterial and 21 (5%) of these fungal OTUs weredetected. In roots of plants grown in the Idaho field (Tables 29, 30),364 (13%) of these bacterial and 14 (3%) of these fungal OTUs weredetected. In roots of plants grown in the Minnesota field (Tables 31,32), 335 (12%) of these bacterial and 13 (3%) of these fungal OTUs weredetected.

Among all the previously detected seed OTUs, 68 bacterial OTUs and 27fungal OTUs were found to be core taxa across crops (Tables 13, 14).Searching for these core OTUs in roots of plants grown in theMassachusetts greenhouse (Tables 25, 26), 27 (40%) of the bacterial and6 (22%) of the fungal OTUs were detected. In roots of plants grown inthe Texas greenhouse (Tables 27, 28), 38 (56%) of these core bacterialand 5 (19%) of these core fungal OTUs were detected. In roots of plantsgrown in the Idaho field (Tables 29, 30), 24 (35%) of these corebacterial and 5 (19%) of these core fungal OTUs were detected. In rootsof plants grown in the Minnesota field (Tables 31, 32), 36 (53%) ofthese core bacterial and 6 (22%) of these core fungal OTUs weredetected. Searching for core seed OTUs that occurred in at least oneroot sample of the greenhouses and fields, 49 (72%) of bacterial and 7(26%) of fungal OTUs were detected. Among these, the most commonbacterial seed OTUs also observed in roots were B0.9|GG99|128181(observed in 100% of all samples), B0.9|GG99|73880 (observed in 96% ofall samples) B0.9|GG99|25580 (observed in 90% of all samples) andB0.9|GG99|132333 (observed in 84% of all samples). Among fungal OTUsfrom seeds, the most commonly observed in roots were F0.9|UDYN|206476(observed in 76% of all samples), F0.9|UDYN|212600 (observed in 46% ofall samples) F0.9|UDYN|216250 (observed in 42% of all samples) andF0.9|UDYN|215392 (observed in 42% of all samples). The fungal SYMStrains 15926, 15928, 00120, 00880, 01325, 01326, 01328, and 15811 allhave greater than 97% identity to OTU F0.9|UDYN|206476 and were assayedon seedlings. The fungal SYM Strains 00741b, 01315, 01327, and 15890 allhave greater than 97% identity to OTU F0.9|UDYN|212600 and were assayedon seedlings. The fungal SYM Strains 00154, 15825, 15828, 15837, 15839,15870, 15872, 15901, 15920, 15932, and 15939 all have greater than 97%identity to F0.9|UDYN|215392 and were assayed on seedlings. The coreseed and root bacteria observed in these experiments belong to the PhylaAcidobacteria, Actinobacteria, Bacteroidetes, Firmicutes,Proteobacteria, and Tenericutes, while the fungal belong to the PhylaAscomycota and to the of the Classes Dothideomycetes andSordariomycetes, which means these groups poses the capacity to be seedtransmitted and to colonize different life stages of a plant (both seedsand roots), as well as having a broad host range and flexibilityallowing them to exist within different species of plant.

29 seed bacterial OTUs were observed in roots of plants grown on sterilesand, while 43 seed bacterial OTUs were observed in roots of plantsgrown in soil; 23 of the same seed OTUs were observed in both conditions(Tables 25, 26). This pattern means that the soil condition enhancescolonization of seed transmitted bacteria into the root. A contrastingtrend was observed for fungi, where 28 seed bacterial OTUs were observedin roots of plants grown on sterile sand, while 19 seed bacterial OTUswere observed in roots of plants grown in soil; 15 of the same seed OTUswere observed in both conditions. Unlike bacteria, seed transmittedfungi attempting to colonize roots growing in non-sterile soil facegreater competition for the root niche as soil transmitted fungi attemptto colonize the root.

Some seed endophytes are especially robust and able to colonize roots ofplants growing in different field and greenhouse environments. Bycounting OTUs of microbes occurring in at least one plant variety ineach field and the Texas greenhouse, 163 bacterial OTUs (out of a totalof 583 bacterial seed OTUs detected in field grown roots) and 8 fungalOTUs (out of a total of 28 fungal seed OTUs detected in roots) wereobserved occurring in roots of these plants in all three environments;these sequences represent robust root colonizers which persist in rootsdespite different environmental conditions.

Root microbiomes in both environments shared 45% of their bacterial seedOTUs (216 OTUs) and 50% of their fungal seed OTUs (9 OTUs). As theseplants were harvested when they were two weeks old, approximately halfthe diversity of seed transmitted microbiomes in maize, wheat and soyseeds are able to colonize and persist in seedlings under agronomicallyrelevant conditions for at least two weeks.

Among all the previously detected seed OTUs found in corn seeds, 20bacterial OTUs and 3 fungal OTUs were found to be present only seeds ofwild and ancient landraces, but not modern varieties of corn. Searchingfor these in roots of ancestral maize varieties grown on sterile sand inthe Massachusetts greenhouse, bacterial OTUs B0.9|GG99|813062,B0.9|GG99|9943, and B0.9|GG99|4327501 (but no fungal OTUs) were detectedin wild maize, and bacterial OTU B0.9|GG99|9943 and fungal OTUF0.9|UDYN|210204 was detected in ancient landrace maize (Tables 25, 26).No ancestral seed bacterial OTU was observed in roots of wild orlandrace maize plants grown on soil in the Massachusetts greenhouse,however fungal OTU F0.9|UDYN|210204 was observed in the roots of theancient landrace maize growing on soil (Tables 25, 26).

Example 3 Isolation of Bacterial Endophytes from Ancestral Seeds

In order to better understand the role played by seed-derived endophyticmicrobes from ancestral species of plants in improving the vigor,general health and stress resilience of modern host plants, we initiateda systematic screen to isolate and characterize endophytic microbes fromseeds of commercially significant grass plants.

Diverse types of wild relatives or ancestral landraces of maize, wheat,rice, and other seeds were acquired and screened for cultivatablemicrobes.

Pools of 5 seeds were soaked in 10 mL of sterile water contained insterile 15 mL conical tubes for 24 hours. Some maize and rice accessionswere sampled for seed surface microbes. In these cases, after 24 hoursof soaking, 50 μL aliquots of undiluted, 100× dilute and 10000× dilutesoaking water was plated onto R2A agar [Proteose peptone (0.5 g/L),Casamino acids (0.5 g/L), Yeast extract (0.5 g/L), Dextrose (0.5 g/L)Soluble starch (0.5 g/L), Dipotassium phosphate (0.3 g/L), Magnesiumsulfate 7H₂O (0.05 g/L), Sodium pyruvate (0.3 g/L), Agar (15 g/L), FinalpH 7±0.2 @ 25° C.] to culture oligotrophic bacteria, while the samevolumes and dilutions were also plated onto potato dextrose agar (PDA)[Potato Infusion from 200 g/L, Dextrose 20 g/L, Agar 15 g/L, Final pH:5.6±0.2 at 25° C.] to culture copiotrophic bacteria and fungi. All seedsin the study were sampled for endophytes by surface sterilization,trituration, and culturing of the mash. Seeds were surface sterilized bywashing with 70% EtOH, rinsing with water, then washing with a 3%solution of sodium hypochlorite followed by 3 rinses in sterile water.All wash and rinse steps were 5 minutes with constant shaking at 130rpm. Seeds were then blotted on R2A agar which was incubated at 30° C.for 7 days in order to confirm successful surface sterilization.Following the sterilization process, batches of seeds were ground with asterile mortar and pestle in sterile R2A broth, while a select number ofsurface sterilized maize, rice and soy seeds were grown in sterileconditions and the roots or shoots of seedlings (without furthersterilization) were crushed by bead beating in a Fastprep24 machine with3 carbide beads, 1 mL of R2A in a 15 mL Falcon tube shaking at 6M/s for60 seconds. Extracts of surface washes, crushed seed, or maceratedseedling tissue were serially diluted by factors of 1 to 10⁻³ and spreadonto quadrants on R2A and PDA agar in order to isolate cultivableseed-borne microorganisms. Plates were incubated at 28° C. for 7 days,monitoring for the appearance of colonies daily. After a week, plateswere photographed and different morphotypes of colonies were identifiedand labeled. These were then selected for identification by sequencing,backing up as glycerol stock, and assaying for beneficial functions asdescribed herein.

Plating and Scoring of Microbes

After 7 days of growth, most microbial colonies had grown large anddistinct enough to allow differentiation based on colony size, shape,color and texture. Photographs of each plate were taken, and on thebasis of color and morphotype, different colonies were identified bynumber for later reference. These strains were also streaked out ontoeither R2A or PDA to check for purity, and clean cultures were thenscraped with a loop off the plate, resuspended in a mixture of R2A andglycerol, and frozen away in quadruplicate at −80° C. for later.

Sequence Analysis & Phylogenetic Assignment of Microbes Isolated fromAncestral Seeds

To accurately characterize the isolated microbial endophytes, colonieswere submitted for marker gene sequencing, and the sequences wereanalyzed to provide taxonomic classifications. Colonies were subjectedto 16S rRNA gene PCR amplification using the 27f/1492r primer set, andSanger sequencing of paired ends was performed at Genewiz (SouthPlainfield, N.J.). Raw chromatograms were converted to sequences, andcorresponding quality scores were assigned using TraceTuner v3.0.6beta(U.S. Pat. No. 6,681,186, incorporated herein by reference). Thesesequences were quality filtered using PRINSEQ v0.20.3 [Schmieder andEdwards (2011) Bioinformatics. 2011; 27:863-864, incorporated herein byreference] with left and right trim quality score thresholds of 30 and aquality window of 20 bp. Sequences without paired reads were discardedfrom further processing. Paired end quality filtered sequences weremerged using USEARCH v7.0 [Edgar (2010) Nature methods 10:996-8].Taxonomic classifications were assigned to the sequences using the RDPclassifier [Wang et al., (2007) Applied and environmental microbiology73:5261-7, incorporated herein by reference] trained on the Greengenesdatabase [McDonald et al. (2012), ISME journal 6:610-8, incorporatedherein by reference]. The resulting 253 microbes, derived from ancestral(wild or ancient landraces) representing over 41 distinct OTUs (using a97% similarity threshold) are provided in Table 24.

Example 4 Characterization of Bacterial Endophytes Isolated fromAncestral Seeds

A total of 140 seed-origin bacterial endophytes were seeded onto 96 wellplates and tested for various activities and/or production of compounds,as described below. The results of these in vitro assays are summarizedin Table 31.

TABLE 31 (Summnary of in vitro characterization of bacterial endophytesisolated from ancestral seeds) Shows SEQ Antag- Antag- Cellulo- SecretesPhosphate Growth on ACC Produces Sym ID onize onize lytic sidero-Solubi- Nitrogen Deaminase Auxin/ Produces Strain ID Source NO: E. coliS. cerevisae activity phores lization Free LGI Activity Indoles AcetoinSYM00033 Wild relative 3117 0 0 1 1 2 No 0 3 0 SYM00620 Wild relative3159 0 1 1 0 1 No 0 2 2 SYM00176 Wild relative 3154 1 0 1 2 1 No 0 2 1SYM00658 Wild relative 3139 1 1 1 0 2 No 1 2 3 SYM00660 Wild relative3127 0 1 2 1 0 No 1 0 1 SYM00011 Wild relative 3123 0 0 0 0 1 Yes 0 2 0SYM00011b Wild relative 3245 0 0 0 0 0 No 0 0 1 SYM00069 Wild relative3232 0 0 0 0 0 No 0 0 2 SYM00013 Wild relative 3160 0 0 2 2 0 Yes 0 2 0SYM00014 Wild relative 3165 0 0 2 1 0 Yes 0 2 0 SYM00062 Wild relative3155 0 0 2 2 0 No 1 2 0 SYM00068 Wild relative 3140 0 0 2 2 1 No 3 2 0SYM00657 Wild relative 3156 0 0 2 0 0 No 3 2 0 SYM00672 Wild relative3144 0 0 2 2 1 No 3 1 0 SYM00178 Ancient Landrace 3196 0 0 1 1 0 No 0 01 SYM00722 Ancient Landrace 3197 0 0 1 0 0 No 1 1 0 SYM00013b Wildrelative 3246 0 0 0 0 0 No 0 0 1 SYM00180 Ancient Landrace 3247 0 0 0 00 No 0 0 1 SYM00181 Ancient Landrace 3233 0 0 0 0 0 No 0 0 2 SYM00525Wild relative 3218 0 0 0 0 0 No 0 2 1 SYM00716 Ancient Landrace 3219 0 00 0 0 No 1 1 1 SYM00731B Ancient Landrace 3234 0 0 0 0 0 No 1 1 0SYM00597 Ancient Landrace 3198 0 0 0 0 1 No 0 0 3 SYM00022 Wild relative3181 0 0 1 1 0 No 0 2 0 SYM00025 Ancient Landrace 3182 0 0 1 0 0 No 0 21 SYM00047 Ancient Landrace 3172 0 0 1 0 2 No 0 1 1 SYM00055 AncientLandrace 3183 0 0 1 1 2 No 0 0 0 SYM00081 Ancient Landrace 3173 0 0 1 12 Yes 0 1 0 SYM00094 Ancient Landrace 3166 0 0 1 1 2 Yes 0 1 1 SYM00095Ancient Landrace 3167 0 0 1 1 2 Yes 0 1 1 SYM00096 Ancient Landrace 31840 0 1 1 0 No 0 1 1 SYM00506 Ancient Landrace 3161 0 0 1 1 1 No 0 3 1SYM00018 Ancient Landrace 3235 0 0 0 0 0 No 0 2 0 SYM00020 AncientLandrace 3199 0 0 0 0 1 Yes 0 3 0 SYM00506b Ancient Landrace 3145 0 1 11 1 No 0 3 3 SYM00731A Ancient Landrace 3174 0 0 1 0 1 No 1 2 0 SYM00049Ancient Landrace 3116 0 0 0 1 0 No 0 3 1 SYM00057 Ancient Landrace 32480 0 0 0 0 No 0 0 1 SYM00058 Ancient Landrace 3220 0 0 0 0 0 No 0 0 3SYM00082a Ancient Landrace 3236 0 0 0 1 0 Yes 0 1 0 SYM00101 AncientLandrace 3221 0 0 0 1 0 No 0 2 0 SYM00502 Ancient Landrace 3185 0 0 0 11 No 0 3 0 SYM00511 Ancient Landrace 3222 0 0 0 0 0 No 0 2 1 SYM00514bAncient Landrace 3162 0 0 0 0 2 No 0 3 3 SYM00514C Ancient Landrace 32000 0 0 0 0 No 3 0 1 SYM00514D Ancient Landrace 3186 0 0 0 0 0 No 0 2 3SYM00100 Ancient Landrace 3157 1 1 1 1 1 No 0 3 0 SYM00078 AncientLandrace 3141 3 1 1 1 2 Yes 0 3 0 SYM00544 Ancient Landrace 3187 0 1 0 01 No 0 3 0 SYM00545B Ancient Landrace 3223 0 1 0 0 0 No 0 2 0 SYM00548Ancient Landrace 3201 0 1 0 0 1 No 0 2 0 SYM00552 Ancient Landrace 32020 1 0 0 0 No 0 2 1 SYM00558 Ancient Landrace 3203 0 1 0 0 1 No 0 2 0SYM00583 Ancient Landrace 3204 0 1 0 0 1 No 0 2 0 SYM00584 AncientLandrace 3224 0 0 0 0 1 No 0 2 0 SYM00588 Ancient Landrace 3168 0 1 0 02 No 0 2 2 SYM00596 Ancient Landrace 3114 0 1 0 0 1 No 0 2 3 SYM00600Ancient Landrace 3188 0 1 0 0 2 No 0 2 0 SYM00746 Ancient Landrace 31751 1 0 0 1 No 1 1 1 SYM00064a Wild relative 3142 0 0 0 0 0 No 0 1 0SYM00183 Wild relative 3176 0 0 0 0 0 No 0 1 2 SYM00184 Wild relative3205 0 0 0 0 0 No 0 1 3 SYM00543 Ancient Landrace 3225 1 1 0 0 0 No 0 10 SYM00595 Ancient Landrace 3118 1 1 0 0 0 No 0 1 0 SYM00551 AncientLandrace 3189 0 1 0 1 0 No 2 1 0 SYM00547 Ancient Landrace 3129 0 0 0 20 No 1 1 0 SYM00560 Ancient Landrace 3226 0 0 0 1 0 No 0 2 0 SYM00586bAncient Landrace 3190 0 1 0 2 0 No 0 2 0 SYM00585 Ancient Landrace 31770 0 0 1 2 No 1 2 0 SYM00824 Ancient Landrace 3192 0 1 0 0 0 No 3 1 0SYM00588b Ancient Landrace 3191 0 0 0 0 0 No 0 3 2 SYM00591 AncientLandrace 3206 0 0 0 0 0 No 3 1 0 SYM00828 Ancient Landrace 3237 0 0 0 10 No 0 1 0 SYM00830 Ancient Landrace 3207 0 0 0 0 0 No 3 1 0 SYM00831Ancient Landrace 3208 0 0 0 1 1 No 1 1 0 SYM00052 Wild relative 3133 0 01 0 1 No 0 1 1 SYM00053 Wild relative 3209 0 0 1 0 1 No 0 0 1 SYM00054Wild relative 3210 0 0 0 1 0 No 0 0 3 SYM00028 Ancient Landrace 3115 1 11 0 1 No 0 1 3 SYM00633 Ancient Landrace 3138 1 1 1 0 2 No 1 3 3SYM00538E Ancient Landrace 3158 1 1 0 2 1 No 3 1 0 SYM00574 AncientLandrace 3149 2 1 0 2 1 No 3 1 1 SYM00501 Ancient Landrace 3146 3 1 0 20 No 3 2 0 SYM00504 Ancient Landrace 3147 3 1 0 2 0 No 3 2 0 SYM00536Ancient Landrace 3148 3 1 0 3 1 No 1 2 0 SYM00575 Ancient Landrace 31503 1 0 2 1 No 3 1 0 SYM00542 Ancient Landrace 3214 0 0 1 0 0 No 0 1 1SYM00556 Ancient Landrace 3193 0 0 1 0 0 No 0 3 0 SYM00586c AncientLandrace 3178 0 0 1 0 0 No 0 2 2 SYM00177 Wild relative 3211 0 0 0 0 0No 0 1 3 SYM00514A Ancient Landrace 3212 0 0 0 0 0 No 0 2 2 SYM00523Wild relative 3213 0 0 0 0 0 No 0 2 2 SYM00538H Ancient Landrace 3238 00 0 0 0 No 0 0 2 SYM00598 Ancient Landrace 3227 0 0 0 0 0 No 0 1 2SYM00051 Wild relative 3163 0 2 0 2 0 No 0 2 2 SYM00587 Ancient Landrace3169 0 0 2 0 0 No 0 2 1 SYM00104 Ancient Landrace 3249 1 0 0 0 0 Yes 0 00 SYM00832 Ancient Landrace 3239 1 0 0 0 0 No 0 0 1 SYM00252 AncientLandrace 3485 0 0 0 0 0 Yes 0 0 0 SYM00182 Ancient Landrace 3151 1 0 1 01 No 1 3 3 SYM00179 Ancient Landrace 3164 1 0 2 0 1 No 0 1 1 SYM00021Wild relative 3131 2 0 3 2 0 No 0 2 0 SYM00589 Ancient Landrace 3126 0 00 0 0 No 0 3 2 SYM00057B Ancient Landrace 3113 0 1 1 1 1 Yes 3 1 0SYM00102 Ancient Landrace 3124 0 0 0 0 0 No 0 0 2 SYM00553 AncientLandrace 3240 0 1 0 0 0 No 0 0 1 SYM00601 Ancient Landrace 3215 1 0 0 00 No 0 0 3 SYM00507 Ancient Landrace 3179 2 1 0 0 0 No 0 2 1 SYM00072Wild relative 3194 2 0 0 0 0 No 0 0 3 SYM00564 Ancient Landrace 3228 2 10 0 0 No 0 0 0 SYM00075 Wild relative 3134 2 0 0 0 0 No 0 0 3 SYM00562Ancient Landrace 3241 2 0 0 0 0 No 0 0 0 SYM00062b Wild relative 3180 00 1 0 0 No 0 3 1 SYM00065 Wild relative 3250 0 0 0 0 0 No 0 0 1 SYM00975Ancient Landrace 3128 0 0 0 2 2 No 0 0 3 SYM00545 Ancient Landrace 32290 1 0 0 0 No 0 2 0 SYM00554 Ancient Landrace 3130 0 1 0 0 0 No 0 1 1SYM00555 Ancient Landrace 3252 0 1 0 0 0 No 0 0 0 SYM00506c AncientLandrace 3216 0 0 0 0 0 No 0 3 1 SYM00506D Ancient Landrace 3242 0 0 0 00 No 0 2 0 SYM00549 Ancient Landrace 3251 0 0 0 0 0 No 0 1 0 SYM00012Wild relative 3121 1 0 0 0 1 No 0 1 1 SYM00050 Ancient Landrace 3153 0 21 1 1 No 0 2 2 SYM00046 Ancient Landrace 3136 1 3 1 2 1 No 0 1 3SYM00106 Ancient Landrace 3243 0 0 1 0 0 Yes 0 0 0 SYM00108 AncientLandrace 3244 0 0 1 0 0 Yes 0 0 0 SYM00107 Ancient Landrace 3125 0 0 0 00 Yes 0 0 1 SYM00090 Ancient Landrace 3122 1 0 0 1 0 No 0 0 0 SYM00002Wild relative 3119 0 0 2 0 0 No 0 3 0 SYM00060 Ancient Landrace 3137 0 00 0 0 No 0 0 3 SYM00071 Wild relative 3120 0 0 0 0 0 No 0 0 2 SYM00563Ancient Landrace 3553 0 0 0 0 0 No 0 0 0 SYM00617 Wild relative 3230 0 00 0 0 No 0 1 2 SYM00960 Ancient Landrace 3195 0 0 0 2 0 No 0 0 3SYM00992 Wild relative 3152 0 0 0 0 2 No 0 0 2 SYM00524 Wild relative3217 0 0 0 0 0 No 0 1 3 SYM00063 Wild relative 3170 1 0 0 0 0 No 0 1 3SYM00527 Wild relative 3171 0 0 1 0 1 No 0 3 1 SYM00538A AncientLandrace 3143 0 0 1 0 0 No 0 2 0 SYM00508 Ancient Landrace 3135 0 0 1 01 No 0 2 0

Production of Auxin

Indole containing IAA is able to generate a pinkish chromophore underacidic conditions in the presence of ferric chloride. Microbial strainswere inoculated into R2A both supplemented with L-TRP (5 mM) in 2 mL 96well culture plates (1 mL). The plate was sealed with a breathablemembrane and incubated at 23° C. under static conditions for 5 days.After 5 days, 150 μL of each culture was transferred to a 96 well plateand the OD600 measured. After measuring the OD600, the plate wascentrifuged, and 50 μL of supernatant was transferred to a new 96 wellplate, mixed with 100 μL of Salkowski reagent (1 mL of FeCl3 0.5 Msolution to 50 mL of 35% HClO4) and incubated in the dark for 30 minutesand OD530 nm measured to detect the pink/red color.

Auxin is an important plant hormone, which can promote cell enlargementand inhibit branch development (meristem activity) in above ground planttissues, while below ground it has the opposite effect, promoting rootbranching and growth. Interestingly, plant auxin is manufactured aboveground and transported to the roots. It thus follows that plant andespecially root inhabiting microbes which produce significant amounts ofauxin will be able to promote root branching and development even underconditions where the plant reduces its own production of auxin. Suchconditions can exist for example when soil is flooded and rootsencounter an anoxic environment.

We screened seed derived bacteria for their ability to produce auxins aspossible root growth promoting agents. A very large proportion of thebacteria tested, approximately 103 out of 140, or 73% of the totalstrains, showed a detectable level of pink or red colour development(the diagnostic feature of the assay suggesting auxin or indoliccompound production). 63 strains (45% of total) had particularly strongproduction of auxin or indole compounds.

Mineral Phosphate Solubilization

Microbes were plated on tricalcium phosphate media as described inRodriguez et al., (2001) J Biotechnol 84: 155-161 (incorporated hereinby reference). This was prepared as follows: 10 g/L glucose, 0.373 g/LNH₄NO₃, 0.41 g/L MgSO₄, 0.295 g/L NaCl, 0.003 FeCl₃, 0.7 g/L Ca₃HPO₄,100 mM Tris and 20 g/L Agar, pH 7, then autoclaved and poured intosquare Petri plates. After 3 days of growth at 28° C. in darkness, clearhalos were measured around colonies able to solubilize the tricalciumphosphate.

Approximately 50 strains (36% of the strains), showed some ability tosolubilize mineral phosphate, with 15 strains (11%) producing stronglevels of mineral phosphate solubilization.

Growth on Nitrogen Free LGI Media

All glassware was cleaned with 6 M HCl before media preparation. A new96 well plate (300 ul well volume) was filled with 250 ul/well ofsterile LGI broth [per L, 50 g Sucrose, 0.01 g FeCl₃-6H₂O, 0.02 g CaCl₂,0.8 g K₃PO₄, 0.2 g CaCl2, 0.2 g MgSO₄-7H₂O, 0.002 g Na₂MoO₄-2H₂O, pH7.5]. Microbes were inoculated into the 96 wells simultaneously with aflame-sterilized 96 pin replicator. The plate was sealed with abreathable membrane, incubated at 28° C. without shaking for 3 days, andOD₆₀₀ readings taken with a 96 well plate reader.

A nitrogen fixing plant associated bacterium is able theoretically toadd to the host's nitrogen metabolism, and the most famous beneficialplant associated bacteria, rhizobia, are able to do this withinspecially adapted organs leguminous plant grows for them to be able todo this. These seed associated bacteria may be able to fix nitrogen inassociation with the developing seedling, whether they colonize theplant's surfaces or interior and thereby add to the plant's nitrogennutrition.

In total, of the 140 isolates there were 15 (10% of strains tested)which had detectable growth under nitrogen limiting conditions (Table31).

ACC Deaminase Activity

Microbes were assayed for growth with ACC as their sole source ofnitrogen. Prior to media preparation all glassware was cleaned with 6 MHCl. A 2 M filter sterilized solution of ACC (#1373A, Research Organics,USA) was prepared in water. 2 μl/mL of this was added to autoclaved LGIbroth (see above), and 250 uL aliquots were placed in a brand new(clean) 96 well plate. The plate was inoculated with a 96 pin libraryreplicator, sealed with a breathable membrane, incubated at 28° C.without 3 days, and OD600 readings taken. Only wells that weresignificantly more turbid than their corresponding nitrogen free LGIwells were considered to display ACC deaminase activity.

Plant stress reactions are strongly impacted by the plant's ownproduction and overproduction of the gaseous hormone ethylene. Ethyleneis metabolized from its precursor 1-aminocyclopropane-1-carboxylate(ACC) which can be diverted from ethylene metabolism by microbial andplant enzymes having ACC deaminase activity. As the name implies, ACCdeaminase removes molecular nitrogen from the ethylene precursor,removing it as a substrate for production of the plant stress hormoneand providing for the microbe a source of valuable nitrogen nutrition.It is somewhat surprising, but this microbially mediated biochemicalability to reduce plant stress is very important as damage to plantgrowth under various stress conditions is believed to result from overproduction of ethylene (Journal of Industrial Microbiology &Biotechnology, October 2007, Volume 34, Issue 10, pp 635-648).

In total, of the 140 isolates there were 28 strains (20%) which hadgreater growth on nitrogen free LGI media supplemented with ACC, than innitrogen free LGI. Of these, 14 strains (10%) had very high ACCdeaminase activity.

Acetoin and Diacetyl Production

The method was adapted from Phalip et al., (1994) J Basic Microbiol 34:277-280. (incorporated herein by reference). 250 ml of autoclaved R2Abroth supplemented with 0.5% glucose was aliquoted into a 96 well plate(#07-200-700, Fisher). The microbial endophytes from a glycerol stockplate were inoculated using a flame-sterilized 96 pin replicator, sealedwith a breathable membrane, then incubated for 3 days without shaking at28° C. At day 3, 50 μl/well was added of freshly blended Barritt'sReagents A and B [5 g/L creatine mixed 3:1 (v/v) with freshly prepared∝-naphthol (75 g/L in 2.5 M sodium hydroxide)]. After 15 minutes, plateswere scored for red or pink colouration relative to a copper colourednegative control (measured as 525 nm absorption on a plate reader).

A very high proportion of the stains tested were found to produceacetoin: 76 strains of the 140 tested, or 54%, produced at least somedetectable level of acetoin, with 40 strains (28%) producing moderate tohigh levels (Table 31).

Siderophore Production

To ensure no contaminating iron was carried over from previousexperiments, all glassware was deferrated with 6 M HCl and water priorto media preparation [Cox (1994) Methods Enzymol 235: 315-329,incorporated herein by reference]. In this cleaned glassware, R2A brothmedia, which is iron limited, was prepared and poured (250 ul/well) into96 well plates and the plate then inoculated with microbes using a 96pin plate replicator. After 3 days of incubation at 28° C. withoutshaking, to each well was added 100 ul of O-CAS preparation withoutgelling agent [Perez-Miranda et al. (2007), J Microbiol Methods 70:127-131, incorporated herein by reference]. Again using the cleanedglassware, 1 liter of O-CAS overlay was made by mixing 60.5 mg of Chromeazurol S (CAS), 72.9 mg of hexadecyltrimethyl ammonium bromide (HDTMA),30.24 g of finely crushed Piperazine-1,4-bis-2-ethanesulfonic acid(PIPES) with 10 ml of 1 mM FeCl₃.6H₂O in 10 mM HCl solvent. The PIPEShad to be finely powdered and mixed gently with stirring (not shaking)to avoid producing bubbles, until a dark blue colour was achieved. 15minutes after adding the reagent to each well, colour change was scoredby looking for purple halos (catechol type siderophores) or orangecolonies (hydroxamate siderophores) relative to the deep blue of theO-Cas.

In many environments, iron is a limiting nutrient for growth. A copingmechanism which many microbes have developed is to produce and secreteiron chelating compounds called siderophores which often only thatparticular species or strain has the means to re-uptake and interactwith to release the bound iron, making it available for metabolism. Afringe effect of siderophore production and secretion is that asiderophore secreting microbes can remove all the bio-available iron inits environment, making it difficult for a competing species to invadeand grow in that micro-environment.

Siderophore production by microbes on a plant surface or inside a plantmay both show that a microbe is equipped to grow in a nutrient limitedenvironment, and perhaps protect the plant environment from invasion byother, perhaps undesirable microbes. Siderophore production wasdetectable in 45 strains (32%), with 18 strains producing significantamounts.

Cellulase Activity

Iodine reacts with cellulose to form a dark blue-colored complex,leaving clear halos as evidence of extracellular enzyme activity.Adapting a previous protocol [Kasana et al. (2008), Curr Microbiol 57:503-507, incorporated herein by reference] 0.2% carboxymethylcellulose(CMC) sodium salt (#C5678, Sigma) and 0.1% triton X-100 were added toR2A media, autoclaved and poured into 150 mm plates. Microbes wereinoculated using a 96 pin plate replicator. After 3 days of culturing inthe darkness at 25° C., cellulose activity was visualized by floodingthe plate with Gram's iodine. Positive colonies were surrounded by clearhalos. 47 strains, or approximately 33%, were found to produce cellulaseactivity. Interestingly, 11 strains produced high levels of cellulaseactivity (Table 31).

Antibiosis

Production of antimicrobial compounds from endophytes was testedessentially as described in Johnston-Monje et al., (2012) PLoS ONE 6(6):e20396, which is incorporated herein by reference. Briefly, colonies ofeither E. coli DH5α (gram negative tester), Bacillus subtillus ssp.subtilis (gram positive tester), or yeast strain Saccharomycescerevisiae AH109 (fungal tester) were resuspended in 1 mL LB to an OD600of 0.2, and 30 μL of this was mixed with 30 mL of warm LB agar. Serialdilutions were made and plates poured. Microbes were inoculated ontorectangular plates containing R2A agar using a 96 pin plate replicator,incubated for one day at 28 C and one day at 23 C. Antibiosis was scoredby observing clear halos around endophyte colonies. 30 strains werefound to produce E. coli-antagonistic activity, while 39 strains hadactivity against S. cerevisiae.

Example 5 Testing of Ancestral Seed-Origin Bacterial EndophytePopulations on Plants Experimental Aim

The results shown above demonstrate that many of the endophytic bacteriaderived from ancestral relatives of modern agricultural plants possessactivities that could impart beneficial traits to a plant uponcolonization. First, many of the bacteria described here are capable ofproducing compounds that could be beneficial to the plant, as detectedusing the in vitro assays described above. In addition, severalrepresentative bacteria were tested and found to successfully colonizecorn plants as demonstrated in the example above. The aim of theexperiments in this section addresses the ability of the bacterialendophytes to confer beneficial traits on a host plant. Severaldifferent methods were used to ascertain this. First, plants inoculatedwith bacteria were tested under conditions without any stress todetermine whether the microbe confers an increase in vigor. Second,endophyte-inoculated plants were tested under specific stress conditions(e.g., salt stress, heat stress, water stress, and combinations thereof)to test whether the bacteria confer an increase in tolerance to thesestresses. These growth tests were performed using two different means:using growth assays on water-agar plates, and using growth assays onsterile filter papers.

Experimental Description Surface Sterilization of Seeds

Un-treated maize seeds and wheat seeds were sterilized overnight withchlorine gas as follows: 200 g of seeds were weighed and placed in a 250mL glass bottle. The opened bottle and its cap were placed in adessicator jar in a fume hood. A beaker containing 100 mL of commercialbleach (8.25% sodium hypochlorite) was placed in the dessicator jar.Immediately prior to sealing the jar, 3 mL of concentrated hydrochloricacid (34-37.5%) were carefully added to the bleach. The sterilizationwas left to proceed for 18-24 h. After sterilization, the bottle wasclosed with its sterilized cap, and reopened in a sterile flow hood. Theopened bottle was left in the sterile hood for a couple hours to air outthe seeds and remove chlorine gas leftover. The bottle was then closedand the seeds stored at room temperature in the dark until use.

Water Agar Assays

Bacterial endophytes isolated from seeds as described herein were testedfor their ability to promote plant growth under normal and stressedconditions by inoculating maize and wheat seeds with those endophytesand germinating them on water agar. For each bacterial endophyte tested,5 mL of liquid R2A medium was inoculated with a single colony and theculture grown at room temperature on a shaker to an OD (600 nm) ofbetween 0.8 and 1.2.

Sterilized maize and wheat seeds were placed on water agar plates (1.3%bacto agar) in a laminar flow hood, using forceps previously flamed. Adrop of inoculum with an OD comprised between 0.8 and 1.2 (correspondingto about 10⁸ CFU/mL) was placed on each seed (50 uL for maize, 30 uL forwheat, representing approximately 5.10⁶ and 3.10⁶ CFUs for maize andwheat, respectively). For each treatment, 3 plates were prepared with 12seeds each. Plates were sealed with surgical tape, randomized to avoidposition effects and placed in a growth chamber set at 22° C., 60%relative humidity, in the dark for four days. After four days, a pictureof each plate was taken and the root length of each seedling wasmeasured using the imaging software ImageJ. The percentage differencebetween the treated plants and the mock-treated (R2A control) was thencalculated. For growth under salt stress, the water agar plates weresupplemented with 100 mM NaCl. For growth under heat stress, the plateswere placed at 40° C., 60% humidity after two days of growth, and leftfor an additional two days.

Filter Paper Growth Assay

Filter papers were autoclaved and placed into Petri dishes, and thenpresoaked with treatment solutions. To simulate normal conditions, 3-4mL sterile water was added to the filters. Water and saline stresseswere induced by adding 3-4 mL 8% PEG 6000 solution or 50 or 100 mM NaClto the filter papers. Surface sterilized seeds were incubated inbacterial inocula for at least one hour prior to plating. Nine seedswere plated in triplicate for each condition tested, including roomtemperature and heat stress (40° C.) for both normal and salineconditions. During initial stages of the experiment, plates were sealedwith parafilm to inhibit evaporative water loss and premature drying ofthe filter papers. Plates were incubated in the dark at room temperaturefor two days following which heat treatment plates were shifted to 40°C. for 4-6 days. Parafilm was removed from all plates after 3-5 days.After 5-8 days, seedlings were scored by manually measuring root lengthfor corn and shoot length for wheat and recording the mass of pooledseedlings from individual replicates.

Experimental Results

Plant vigor and improved stress resilience are important components ofproviding fitness to a plant in an agricultural setting. These can bemeasured in germination assays to test the improvement on the plantphenotype as conferred by microbial inoculation. The collection ofseed-derived endophytes produced a measurable response in corn and wheatwhen inoculated as compared to non-inoculated controls, as shown inTables 32A-32D. For example, most of the strains tested were found toproduce a favorable phenotype in any of the measured multiple parameterssuch as root length, weight, or shoot length in wheat, suggesting thatthe strains play an intimate role modulating and improving plant vigorand conferring stress resilience to the host plant. In wheat undernormal conditions (vigor), 78% of the strains tested showed some levelof effect and 63% a strong plant response suggesting the physiology andecological niches of the strain collection can be associated to abeneficial plant role. The stress responses in the strain collection canbe seen by the ability of a subgroup to confer a beneficial responseunder different conditions such as heat and salt and water stress. Thesecan be applicable to products for arid and marginal lands. In a largeproportion of cases for the tested strains, the beneficial effect wasmeasurable in both crops indicating that the strains are capable ofcolonizing multiple varieties and plant species. This can play a role intheir mechanisms for dispersal and colonization from one seed into amature plant but also as part of the life cycle to establish an ampledistribution range and ecological persistence in nature.

TABLE 32A (ancestral seed-origin bacterial endophyte populations onplants in corn assays) Root Root length Weight length Corn CornCorn-variety Old NEW Variety Variety 3 water-agar Strain Source OTU#OTU# 1 2 Normal Salt SYM00002 Wild relative 66 3119 N/A N/A 2 2 SYM00011Wild relative 2 3123 N/A N/A — 1 SYM00012 Wild relative 55 3121 N/A N/A2 2 SYM00028 Ancient 18 3115 N/A N/A 2 N/A Landrace SYM00049 Ancient 73116 2 1 3 1 Landrace SYM00052 Wild relative 18 3133 N/A N/A 1 —SYM00057b Ancient 37 3113 N/A N/A 3 2 Landrace SYM00060 Ancient 67 3137N/A N/A 1 N/A Landrace SYM00064a Wild relative 10 3142 N/A N/A 2 2SYM00071 Wild relative 76 3120 N/A N/A 1 SYM00090 Ancient 62 3122 N/AN/A — 1 Landrace

TABLE 32B (Root length and weight of ancestral seed-origin bacterialendophyte populations on plants) Root length Weight Heat- Water Heat-Water Strain Normal Heat Salt salt stress Normal Heat Salt salt stressSYM00002 1 3 — — 3 2 3 1 — 1 SYM00011 N/A N/A N/A N/A 2 — — — — 2SYM00012 — 1 — — — 2 2 — 2 — SYM00021 — — 3 1 — — — — — — SYM00028 — — —— 3 1 — 2 3 — SYM00033 — 1 3 2 2 1 3 — 2 — SYM00049 1 3 1 2 1 — — — 1 —SYM00052 N/A N/A N/A N/A 2 — — — — 1 SYM00057b 1 1 — 1 1 1 3 1 1 1SYM00060 3 2 1 — — — 2 — — — SYM00063 — — — — — 1 — — — — SYM00071 — 1 23 — 2 1 2 3 — SYM00075 N/A N/A N/A N/A — — — — — 3 SYM00090 2 2 2 — 1 33 1 1 — SYM00102 — 2 3 3 — — 1 — 3 — SYM00107 — 1 — — — 1 — — 3 1SYM00508 — — — — — 1 — — — — SYM00538A 1 1 3 — — — — — 1 — SYM00547 2 13 — 1 1 — — — 1 SYM00554 — 3 — 3 — — 2 — 3 — SYM00589 — 2 3 3 — 1 3 1 3— SYM00595 1 3 2 2 — 1 3 1 3 — SYM00596 1 3 3 3 1 — 3 — 3 — SYM00660 — 21 1 2 — 2 — — 2 SYM00967 — — 3 — 3 1 1 1 — 1 SYM00975 2 — 3 — 3 1 1 — —2 SYM00992 1 — — — 3 — — — — —

TABLE 32C (Root length in normal, heat, and salt stress modes) RootLength Strain Normal Heat Salt SYM00028 3 — 2 SYM00046 3 N/A N/ASYM00049 3 2 2 SYM00057b 3 3 3 SYM00060 2 N/A N/A SYM00090 3 2 1SYM00102 2 — — SYM00107 2 3 — SYM00508 3 3 1 SYM00538A 1 — 1 SYM00547 23 2 SYM00589 — 3 1 SYM00595 — 3 — SYM00596 1 3 1 SYM00965 2 — 1 SYM009672 3 3 SYM00975 1 — 2 SYM00002 3 — 3 SYM00011 3 3 3 SYM00012 3 1 3SYM00021 3 — — SYM00033 3 — 3 SYM00052 1 — 3 SYM00063 1 — — SYM00064a 3— — SYM00071 3 — — SYM00075 3 — — SYM00183 3 — — SYM00660 — — 2 SYM00992— — 3

TABLE 32D (Root length and weight in normal, salt, and water stresses)Shoot length Weight Water Water Strain Normal Salt stress Normal Saltstress SYM00002 — 1 — — 2 — SYM00011 3 1 3 3 — 2 SYM00012 — 2 3 2 — 1SYM00028 — 3 3 — 3 3 SYM00033 3 1 2 — — 1 SYM00049 3 — 3 2 — 2 SYM000521 — 1 3 — — SYM00057b 3 3 1 2 — 3 SYM00064a — 2 2 — — — SYM00071 2 3 3 —3 1 SYM00075 — 1 3 — — 3 SYM00090 — — 3 — — 3 SYM00102 — 3 3 2 3 —SYM00107 1 3 3 2 3 3 SYM00508 — 3 — — 2 — SYM00547 N/A N/A 1 N/A N/A —SYM00554 — 3 — — 3 — SYM00595 1 3 3 2 3 — SYM00596 1 3 3 1 3 2 SYM00660N/A N/A 3 N/A N/A —

Example 6 Identification and Characterization of Culturable Bacterialand Fungal Endophytes Belonging to OTUs Present in Landrace and WildCorn and Wheat Seeds that have been Lost in Modern Corn and Wheat SeedsIsolation and Identification of Culturable Microbes

In order to better understand the role played by landrace and wildseed-derived endophytic microbes in improving the vigor, general healthand stress resilience of modern agricultural plants, we identifiedculturable microbes that belong to the same OTUs as certain microbes ofTables 16-23 that were present in landrace and wild seeds but werepresent at much lower levels in modern wheat or corn. Using the samemethods as in Example 3 and other techniques known in the art, bacterialendophytes were cultured from a variety of plant parts and a variety ofplants. To accurately characterize the isolated microbial endophytes,colonies were submitted for marker gene sequencing, and the sequenceswere analyzed to provide taxonomic classifications. Among the culturedmicrobes, those with at least 97% 16S or ITS sequence similarity tocertain microbes of Tables 16-19 were identified. Those microbes arelisted in Table 33A.

TABLE 33A Cultured bacterial isolates belonging to the same OTUs ascertain bacteria of Tables 16-19 that were present in landrace and wildseeds but were present at much lower levels in modern wheat or corn. SEQID New SEQ ID NO of NO of bacterial Old OTU of NEW OTU of Culturedcultured taxa from bacterial taxa bacterial taxa bacterial bacterialwild or from wild or from wild or isolate isolate landrace seed landraceseed landrace seed SYM00013 3590 33 OTU_7 B0.9|GG99|4327501 SYM000183592 30, 31 OTU_2, B0.9|GG99|9943, OTU_3489 B0.9|GG97|2582263 SYM00021b3594 27 OTU_35 B0.9|GG99|370327 SYM00025 3595 30, 31 OTU_2,B0.9|GG99|9943, OTU_3489 B0.9|GG97|2582263 SYM00028 27 OTU_35B0.9|GG99|370327 SYM00043 3598 30, 31 OTU_2, B0.9|GG99|9943, OTU_3489B0.9|GG97|2582263 SYM00044 3599 27 OTU_35 B0.9|GG99|370327 SYM00050 360026, 28, OTU_3592, B0.9|GG97|816702, 24, 25, OTU_1384, B0.9|GG99|218527,1939, OTU_3629, B0.9|GG97|639627, 1548 OTU_2970, B0.9|GG97|253061,OTU_3153, B0.9|GG97|4374146, OTU_115 B0.9|GG99|625742 SYM00068 3606 33OTU_7 B0.9|GG99|4327501 SYM00074 3608 26, 28, OTU_3592,B0.9|GG97|816702, 24, 25, OTU_1384, B0.9|GG99|218527, 1939, OTU_3629,B0.9|GG97|639627, 1548 OTU_2970, B0.9|GG97|253061, OTU_3153,B0.9|GG97|4374146, OTU_115 B0.9|GG99|625742 SYM00183 3620 37 OTU_83B0.9|GG99|4102407 SYM00184 3621 37 OTU_83 B0.9|GG99|4102407 SYM002193624 18 OTU_38 B0.9|GG99|29974 SYM00506c 3629 16 OTU_24B0.9|GG99|4294649 SYM00508 3631 25 OTU_2970 B0.9|GG97|253061 SYM005453637 16 OTU_24 B0.9|GG99|4294649 SYM00549 3638 16 OTU_24B0.9|GG99|4294649 SYM00617 3645 18 OTU_38 B0.9|GG99|29974 SYM00620 364626, 28, OTU_3592, B0.9|GG97|816702, 24, 25, OTU_1384, B0.9|GG99|218527,1939, OTU_3629, B0.9|GG97|639627, 1548 OTU_2970, B0.9|GG97|253061,OTU_3153, B0.9|GG97|4374146, OTU_115 B0.9|GG99|625742 SYM00646 3651 33OTU_7 B0.9|GG99|4327501 SYM00662 3653 2005  OTU_11 B0.9|GG99|560886SYM00905 3663 37 OTU_83 B0.9|GG99|4102407

Characterization of Culturable Microbes: Auxin, Acetoin and SiderophoreProduction

The culturable microbes belonging to the same OTUs as certain microbesof Tables 16-23 that were present in landrace and wild seeds but werepresent at much lower levels in modern wheat or corn were then seededonto 96 well plants and tested for auxin, acetoin and siderophoreproduction, using the methods described in Example 5 with minormodifications. For auxin measurement, 1 μl of overnight-grown culturesof endophytic bacterial strains were inoculated into 750 μl of R2A brothsupplemented with L-TRP (5 mM) in 2-mL 96 well culture plates. Theplates were sealed with a breathable membrane and incubated at 23° C.with constant shaking at 200 rpm for 4 days. To measure anxin productionby fungal strains, 3 μl of 5-day old liquid fungal cultures wereinoculated into 1 ml R2A broth supplemented with L-TRP (5 mM) in 24-wellculture plates. The plates were sealed with breathable tape andincubated at 23° C. with constant shaking at 130 rpm for 4 days. After 4days, 100 μL of each culture was transferred to a 96 well plate. 25 μLof Salkowski reagent (1 mL of FeCl3 0.5 M solution to 50 mL of 35%HClO4) was added into each well and the plates were incubated in thedark for 30 minutes before taking picture and measuring 540 nmabsorption using the SpectraMax M5 plate reader (Molecular Devices). Foracetoin measurements, microbial strains were cultured as described abovein R2A broth supplemented with 5% glucose. After 4 days, 100 μL of eachculture was transferred to a 96 well plate and mixed with 25 μLBarritt's Reagents (See Example 4) and 525 nm absorption was measured.For siderophore measurements, microbial strains were cultured asdescribed above in R2A broth. The results are presented in Tables 31A.

TABLE 33B Auxin, siderophore, and acetoin production by culturablebacteria belonging to OTUs present in landrace and wild corn and wheatseeds that are present in lower levels in modern corn and wheat seedsSEQ Secretes Produces Produces Strain ID NO. siderophores Auxin/IndolesAcetoin SYM00013 3590 0 1 0 SYM00018 3592 0 3 2 SYM00021b 3594 0 2 3SYM00025 3595 1 3 2 SYM00043 3598 1 3 2 SYM00044 3599 1 1 3 SYM000503600 1 2 3 SYM00068 3606 2 2 0 SYM00074 3608 2 3 0 SYM00183 3620 0 2 1SYM00184 3621 0 2 0 SYM00219 3624 3 2 3 SYM00506c 3629 0 2 2 SYM005083631 0 3 2 SYM00545 3637 2 2 2 SYM00549 3638 2 2 2 SYM00617 3645 1 3 1SYM00620 3646 1 3 0 SYM00646 3651 3 2 3 SYM00662 3653 1 1 1 SYM009053663 3 2 2

In total, a very large proportion of the bacteria strains, 18 out of 21strains tested, were able to utilize Tryptophan supplemented in themedium and showed a detectable level of pink or red color development(the diagnostic feature of the assay suggesting auxin or indoliccompound production). 7 strains (33% of total) had particularly strongproduction of auxin or indole compounds. As for acetoin production, 13out of 21 strains tested showed a detectable level of pink or red color(a proxy of acetoin production). Particularly, 5 of these 13 strains hadstrong production of acetoin. 7 out of 21 strains tested showed adetectable level of siderophore accumulation. Among these 7 strains, 3strains showed very strong accumulation of siderophore.

Characterization of Culturable Microbes: Substrate Use

In addition to determining whether the strains produce auxin, acetoin,and siderophores, the ability of these strains to grow on a variety ofsubstrates was determined. Liquid cultures of microbe were firstsonicated to achieve homogeneity. 1 mL culture of each strain washarvested by centrifugation for 10 minutes at 4500 RPM and subsequentlywashed three times with sterile distilled water to remove any traces ofresidual media. Microbial samples were resuspended in sterile distilledwater to a final OD₅₉₀ of 0.2. Measurements of absorbance were takenusing a SpectraMax M microplate reader (Molecular Devices, Sunnyvale,Calif.).

Sole carbon substrate assays were done using BIOLOG Phenotype MicroArray(PM) 1 and 2A MicroPlates (Hayward, Calif.). An aliquot of eachbacterial cell culture (2.32 mL) were inoculated into 20 mL sterileIF-0a GN/GP Base inoculating fluid (IF-0), 0.24 mL 100× Dye F obtainedfrom BIOLOG, and brought to a final volume of 24 mL with steriledistilled water. Negative control PM1 and PM2A assays were also madesimilarly minus bacterial cells to detect abiotic reactions. An aliquotof fungal culture (0.05 mL) of each strain were inoculated into 23.95 mLFF-IF medium obtained from BIOLOG. Microbial cell suspensions werestirred in order to achieve uniformity. One hundred microliters of themicrobial cell suspension was added per well using a multichannelpipettor to the 96-well BIOLOG PM1 and PM2A MicroPlates that eachcontained 95 carbon sources and one water-only (negative control) well.

MicroPlates were sealed in paper surgical tape (Dynarex, Orangeburg,N.Y.) to prevent plate edge effects, and incubated stationary at 24° C.in an enclosed container for 70 hours. Absorbance at 590 nm was measuredfor all MicroPlates at the end of the incubation period to determinecarbon substrate utilization for each strain and normalized relative tothe negative control (water only) well of each plate (Garland and Mills,1991; Barua et al., 2010; Siemens et al., 2012; Blumenstein et al.,2015). The bacterial assays were also calibrated against the negativecontrol (no cells) PM1 and PM2A MicroPlates data to correct for anybiases introduced by media on the colorimetric analysis (Borglin et al.,2012). Corrected absorbance values that were negative were considered aszero for subsequent analysis (Garland and Mills, 1991; Blumenstein etal., 2015) and a threshold value of 0.1 and above was used to indicatethe ability of a particular microbial strain to use a given carbonsubstrate (Barua et al., 2010; Blumenstein et al., 2015). Additionally,bacterial MicroPlates were visually examined for the irreversibleformation of violet color in wells indicating the reduction of thetetrazolium redox dye to formazan that result from cell respiration(Garland and Mills, 1991). Fungal PM tests were measured as growthassays and visual observation of mycelial growth in each well was made.

The results of these assays are shown in Tables 31B (BIOLOG PM1MicroPlates) and 31 C (BIOLOG PM2A MicroPlates).

TABLE 33C Substrate utilization as determined by BIOLOG PM1 MicroPlatesby culturable bacteria belonging to OTUs present in landrace and wildcorn and wheat seeds that are present in lower levels in modern corn andwheat seeds. Strain/Substrate SYM13 SYM18 SYM183 SYM184 SYM219 SYM43SYM50 SYM508 SYM617 SYM620 SYM68 SYM905 D-Serine NO NO NO NO NO NO YESNO NO NO NO NO D-Glucose-6- NO NO NO NO NO YES YES YES NO YES NO NOPhosphate L-Asparagine NO NO NO NO NO NO NO NO NO NO NO NO L-glutamineNO NO NO NO NO NO NO NO NO NO NO NO Glycyl-L- YES NO NO NO NO YES YES NONO NO NO NO Aspartic acid Glycyl-L- NO NO YES YES NO NO NO NO NO NO YESNO Glutamic acid Glycyl-L- NO NO YES YES NO NO YES NO NO NO YES YESProline L-Arabinose YES YES NO YES NO YES YES NO NO NO YES NO D-SorbitolNO NO NO YES NO NO YES NO NO NO NO NO D-Galactonic YES YES NO NO YES YESNO NO NO NO NO NO acid-?- lactone D-Aspartic acid NO NO NO NO NO NO NONO NO NO NO NO m-Tartaric acid YES YES NO NO NO YES NO NO NO NO NO NOCitric acid NO NO NO NO NO NO NO NO NO NO YES NO Tricarballylic NO NO NONO NO NO NO NO NO NO NO NO acid p-Hydroxy NO NO NO NO NO NO YES NO NO NONO NO Phenyl acetic acid N-Acetyl-D- YES YES YES YES YES YES YES NO NONO NO NO Glucosamine Glycerol NO NO NO NO NO YES YES NO NO NO NO NOD-L-Malic acid NO NO NO YES NO YES NO YES YES NO YES NO D-GlucosaminicNO YES NO NO NO YES NO YES NO NO NO NO acid D-Glucose-1- NO YES NO NO NOYES YES YES NO NO NO NO Phosphate m-Inositol NO YES NO YES NO YES YES NONO NO NO NO L-Serine NO NO NO NO NO NO NO NO NO NO NO NO m-Hydroxy NO NONO NO NO NO YES NO NO YES NO NO Phenyl Acetic acid D-Saccharic NO NO NOYES NO YES YES YES NO NO NO NO acid L-Fucose NO NO NO NO NO NO NO NO NONO NO NO D-Ribose NO YES YES YES NO YES NO NO NO NO YES NO1,2-Propanediol NO NO NO NO NO NO NO NO NO NO NO NO D-Fructose- NO YESNO NO NO NO YES YES NO YES NO NO 6-Phosphate D-Threonine NO NO NO NO NONO NO NO NO NO NO NO L-Threonine NO NO NO NO NO NO NO NO NO NO NO NOTyramine YES YES YES NO YES NO NO NO NO NO YES NO Succinic acid NO NO NONO NO NO NO NO NO NO NO NO D-Glucuronic NO NO NO NO NO NO YES NO NO NONO NO acid Tween 20 NO NO NO YES NO NO NO NO NO NO NO NO Tween 40 NO NONO NO NO YES NO NO NO NO NO NO Tween 80 NO NO YES YES NO NO NO NO NO NONO YES Fumaric acid NO NO NO NO NO NO NO NO NO NO NO NO L-Alanine YESYES YES YES YES YES YES NO NO YES YES YES D-Psicose NO YES NO NO NO YESNO NO NO NO NO NO D-Galactose YES YES NO YES YES YES YES YES NO NO NO NOD-Gluconic acid NO YES NO NO NO YES YES YES NO YES NO NO L-Rhamnose NOYES NO NO YES YES YES YES YES YES YES NO a-Keto-Glutaric NO NO YES NO NONO YES NO NO NO YES NO acid a-Hydroxy YES NO NO YES NO NO YES NO NO NOYES NO Glutaric acid-?- lactone Bromo NO NO NO NO NO NO NO NO NO NO NONO succinic acid L-Alanyl- YES YES YES YES NO NO YES NO NO NO YES NOGlycine L-Lyxose NO YES NO NO NO YES YES YES NO NO YES NO L-Asparticacid NO NO YES NO NO NO YES YES NO NO NO NO D-L-a-Glycerol NO NO NO NONO NO NO NO NO NO NO NO phosphate D-Fructose NO NO NO YES NO YES YES NONO NO YES NO a-Keto-Butyric NO NO NO NO NO NO NO NO NO NO NO NO acida-Hydroxy NO NO NO NO NO NO NO NO NO NO NO NO Butyric acid Propionicacid NO YES NO NO NO NO NO NO NO NO YES NO Acetoacetic NO NO NO NO NO NONO NO NO NO NO NO acid Glucuronamide NO NO NO NO NO NO NO NO NO NO NO NOL-Proline NO YES YES NO NO NO NO NO NO NO YES NO D-Xylose YES YES YESYES NO YES YES NO NO NO YES NO Acetic acid NO YES NO YES NO YES NO NO NONO NO NO a-Methyl-D- NO NO NO NO YES NO YES NO NO YES NO NO Galactosideβ-Methyl-D- NO YES NO YES YES YES YES YES NO NO NO NO glucoside Muckacid YES YES YES NO NO YES YES YES NO NO YES NO N-acetyl-β-D- NO NO NONO NO NO YES NO YES NO NO NO Mannosamine Pyruvic acid NO YES YES YES YESYES YES YES NO NO YES NO D-Alanine YES YES YES YES YES NO NO NO NO NO NONO L-Lactic acid NO NO NO NO NO YES YES NO NO NO NO NO a-D-Glucose NOYES YES YES NO YES YES NO YES NO NO NO a-D-Lactose NO NO YES YES NO NONO NO NO NO NO NO Adonitol NO YES YES NO NO NO NO NO NO NO NO NOGlycolic acid NO NO NO NO NO NO NO NO NO NO NO NO Mono Methyl NO NO NONO NO NO NO NO NO NO NO NO Succinate L-Galactonic- NO YES YES YES YESYES YES YES NO YES YES NO acid-?- lactone D-Trehalose NO NO NO NO NO YESYES NO NO NO NO NO Formic acid NO YES NO NO NO YES NO NO NO NO NO NOMaltose NO YES YES YES YES YES YES YES YES NO NO YES Lactulose NO NO YESYES NO NO NO NO NO NO NO NO Maltotriose NO YES YES YES YES YES YES YESYES NO NO YES Glyoxylic acid NO NO NO NO NO NO NO NO NO NO NO NO MethylPyruvate NO NO NO NO NO NO YES YES NO NO YES NO D-Galacturonic NO NO YESNO NO YES YES NO NO YES NO NO acid D-Mannose NO YES YES YES NO YES YESNO NO NO NO YES D-Mannitol NO YES NO YES NO YES YES NO NO NO NO NOD-Melibiose NO YES YES YES YES YES YES NO YES NO NO NO Sucrose NO NO YESYES NO YES YES NO NO NO NO NO 2-Deoxy NO YES NO NO NO YES YES YES NO YESNO NO adenosine D-Cellobiose NO YES YES YES YES YES YES YES YES NO NOYES D-Malic acid NO NO NO NO NO NO NO NO NO NO YES NO Phenylethyl- NO NONO NO NO NO NO NO NO NO NO NO amine Dulcitol NO NO NO NO YES YES NO NOYES NO NO NO L-Glutamic NO NO NO NO NO NO YES NO NO NO NO NO acidThymidine NO YES NO NO NO YES YES YES NO NO NO NO Uridine YES YES YESYES NO NO YES YES NO NO NO NO Adenosine NO YES NO NO YES YES NO YES NOYES NO NO Inosine NO NO NO YES NO NO NO NO NO NO NO NO L-Malic acid NONO NO NO NO NO NO NO NO NO NO NO 2-Aminoethanol NO YES YES YES NO NO NONO NO NO NO NO

TABLE 33D Substrate utilization as determined by BIOLOG PM2A MicroPlatesby culturable bacteria belonging to OTUs present in landrace and wildcorn and wheat seeds that are present in lower levels in modern corn andwheat seeds. Strain/Substrate SYM13 SYM18 SYM183 SYM184 SYM219 SYM43SYM50 SYM508 SYM617 SYM620 SYM68 SYM905 N-acetyl-D- NO NO YES YES NO NOYES NO NO NO NO YES Galactosamine Gentiobiose NO YES YES YES YES YES YESYES YES YES NO YES D-Raffinose NO NO NO NO YES NO YES NO NO YES NO NOCapric acid NO NO NO NO NO NO NO NO NO NO NO NO D-lactic acid NO NO NONO NO NO YES NO NO NO NO NO methyl ester Acetamide NO NO NO NO NO NO NONO NO NO YES NO L-Ornithine YES YES NO YES YES NO YES NO NO NO YES NOChondrointin NO NO NO NO NO NO NO NO NO NO NO NO sulfate C N-acetyl- NONO NO NO NO NO NO NO NO YES NO NO neuraminic acid L-glucose NO NO NO NONO NO NO NO NO NO NO NO Salicin NO NO YES YES YES NO YES YES YES NO NOYES Caproic acid NO NO NO NO NO NO NO YES NO NO NO NO Malonic acid NO NONO NO NO NO NO NO NO NO NO NO L-Alaninamide NO NO YES YES NO NO NO NO NONO NO YES L-Phenylalanine YES NO NO NO NO NO NO NO NO NO YES NOa-Cyclodextrin NO NO NO NO NO NO NO NO NO NO NO NO β-D-allose NO NO NONO NO NO NO NO NO NO NO NO Lactitol NO NO YES YES NO NO NO NO NO NO NOYES Sedoheptulosan NO NO NO NO NO NO NO NO NO NO NO NO Citraconic acidYES NO NO NO NO NO NO NO NO NO YES NO Melibionic acid NO NO NO NO YES NOYES NO NO YES YES NO N-Acetyl-L- NO NO NO YES NO NO YES NO NO NO NO NOGlutamic acid L-Pyroglutamic YES YES YES YES YES NO NO YES NO NO YES NOacid β-Cyclodextrin NO NO NO NO YES NO NO NO NO NO NO NO Amygdalin NO NOYES YES NO NO NO NO YES NO NO NO D-Melezitose NO NO NO NO NO NO NO NOYES NO NO NO L-Sorbose NO NO NO NO NO NO NO NO NO NO NO NO Citramalicacid NO NO NO NO NO NO NO NO NO NO NO NO Oxalic acid NO NO NO NO NO NONO NO NO NO NO NO L-Arginine NO NO NO NO NO NO NO NO NO NO NO NOL-Valine YES YES NO YES YES NO NO NO NO NO YES NO γ-Cyclodextrin NO NONO NO YES NO NO NO NO NO NO NO D-arabinose NO NO NO NO NO NO NO YES NONO NO NO Maltitol NO NO YES YES NO NO NO NO NO NO NO YES Stachyose NO NONO NO NO NO NO NO NO NO NO NO D-Glucosamine YES YES YES YES YES YES YESYES YES NO YES YES Oxalomalic acid YES YES YES YES NO YES NO NO YES NOYES YES Glycine NO NO NO NO NO NO NO NO NO NO NO NO D,L-Carnitine YESYES NO NO NO NO NO NO NO NO NO NO Dextrin NO NO NO YES YES NO NO YES YESYES NO NO D-arabitol NO NO NO NO NO NO NO YES NO NO NO NO a-Methyl-D- NONO NO NO NO NO NO NO NO NO NO NO Glucoside D-Tagatose NO NO NO NO NO NONO YES NO NO NO NO 2-Hydroxy NO NO NO NO NO NO NO NO NO NO NO NO benzoicacid Quinic acid NO NO NO NO NO NO NO NO NO NO NO NO L-Histidine NO NONO NO NO YES NO NO NO YES NO NO Sec-Butylamine NO NO NO NO NO NO NO NONO NO NO NO Gelatin NO NO YES YES NO NO NO NO NO NO NO YES L-arabitol NONO NO NO NO NO NO NO NO NO NO NO β-Methyl-D- NO NO NO YES NO NO NO YESNO YES NO NO Galactoside Turanose NO NO YES YES NO NO NO NO NO NO NO NO4-Hydroxy NO NO NO NO NO NO NO NO NO NO NO NO benzoic acid D-Ribono- NONO NO NO NO NO NO NO NO NO NO NO 1,4-Lactone L-Homoserine NO NO NO NO NONO NO NO NO NO NO NO D,L-Octopamine YES YES YES YES YES NO NO NO YES NOYES YES Glycogen NO NO NO NO NO NO NO YES NO NO NO NO Arbutin NO NO YESYES YES NO YES YES YES NO NO YES 3-Methyl Glucose NO NO NO NO NO NO NOYES NO NO NO NO Xylitol NO NO NO YES NO NO NO NO NO NO NO NO β-HydroxyNO NO NO NO NO NO YES YES NO NO NO NO butyric acid Sebacic acid NO NO NONO NO NO NO NO NO NO NO NO Hydroxy-L- NO NO NO NO NO NO YES NO NO NO NONO Proline Putrescine NO YES NO NO NO NO YES NO NO NO NO NO Inulin NO NOYES YES YES YES NO NO NO NO NO NO 2-Deoxy-D- NO NO NO NO NO NO NO YES NOYES NO NO Ribose β-Methyl-D- NO NO NO NO NO NO NO NO NO NO NO NOGlucuronic acid N-Acetyl-D- NO NO NO NO NO NO NO NO NO NO NO NOglucosaminitol γ-Hydroxy NO NO NO NO NO NO NO NO NO NO NO NO butyricacid Sorbic acid NO NO NO NO NO NO NO NO NO NO NO NO L-Isoleucine YES NONO NO NO NO NO YES NO NO YES NO Dihydroxy NO NO NO YES NO NO YES YES NONO NO NO acetone Laminarin NO NO NO NO NO NO NO NO NO NO NO NOi-Erythritol NO NO NO NO NO NO NO NO NO NO NO NO a-Methyl-D- NO NO NO NONO NO NO NO NO NO NO NO Mannoside γ-amino YES YES NO NO NO YES NO NO NONO NO NO butyric acid a-Keto-valeric NO NO NO NO NO NO NO NO NO NO NO NOacid Succinamic acid NO NO NO NO NO NO NO NO NO NO NO NO L-Leucine YESNO NO NO NO NO NO NO NO NO NO NO 2,3-Butanediol YES NO NO NO NO NO NO NONO NO NO NO Mannan NO NO NO NO NO NO NO NO NO NO NO NO D-Fucose NO NO NONO NO NO NO NO NO NO NO NO β-Methyl-D- NO NO NO NO NO NO NO NO NO NO NONO Xyloside d-amino NO NO NO NO NO NO NO NO NO NO NO NO valeric acidItaconic acid NO NO NO NO NO NO YES YES NO NO NO NO D-Tartaric acid NONO NO NO NO NO NO NO NO NO NO NO L-Lysine NO NO NO NO NO NO NO NO NO NONO NO 2,3-Butanone NO NO NO NO NO NO NO NO NO NO NO NO Pectin NO NO NONO NO NO NO YES NO NO NO NO 3-0-β-D- NO NO NO NO NO NO NO NO NO NO NO NOGalactopyranosyl- D-arabinose Palatinose NO NO YES YES YES NO NO NO NONO NO YES Butyric acid NO NO NO NO NO NO NO NO NO NO NO NO 5-Keto-D- NOYES NO NO NO YES NO YES NO NO NO NO Gluconic acid L-Tartaric acid YESYES NO NO NO YES NO YES NO NO NO NO L-Methionine NO NO NO NO NO NO NO NONO NO NO NO 3-Hydroxy 2- NO NO NO NO NO NO NO NO NO NO NO NO ButanoneN-acetyl-D- NO NO YES YES NO NO YES NO NO NO NO YES GalactosamineGentiobiose NO YES YES YES YES YES YES YES YES YES NO YES D-Raffinose NONO NO NO YES NO YES NO NO YES NO NO Capric acid NO NO NO NO NO NO NO NONO NO NO NO D-lactic acid NO NO NO NO NO NO YES NO NO NO NO NO methylester Acetamide NO NO NO NO NO NO NO NO NO NO YES NO L-Ornithine YES YESNO YES YES NO YES NO NO NO YES NO Chondrointin NO NO NO NO NO NO NO NONO NO NO NO sulfate C N-acetyl- NO NO NO NO NO NO NO NO NO YES NO NOneuraminic acid L-glucose NO NO NO NO NO NO NO NO NO NO NO NO Salicin NONO YES YES YES NO YES YES YES NO NO YES Caproic acid NO NO NO NO NO NONO YES NO NO NO NO Malonic acid NO NO NO NO NO NO NO NO NO NO NO NOL-Alaninamide NO NO YES YES NO NO NO NO NO NO NO YES L-Phenylalanine YESNO NO NO NO NO NO NO NO NO YES NO a-Cyclodextrin NO NO NO NO NO NO NO NONO NO NO NO β-D-allose NO NO NO NO NO NO NO NO NO NO NO NO Lactitol NONO YES YES NO NO NO NO NO NO NO YES Sedoheptulosan NO NO NO NO NO NO NONO NO NO NO NO Citraconic acid YES NO NO NO NO NO NO NO NO NO YES NOMelibionic acid NO NO NO NO YES NO YES NO NO YES YES NO N-Acetyl-L- NONO NO YES NO NO YES NO NO NO NO NO Glutamic acid L-Pyroglutamic YES YESYES YES YES NO NO YES NO NO YES NO acid β-Cyclodextrin NO NO NO NO YESNO NO NO NO NO NO NO Amygdalin NO NO YES YES NO NO NO NO YES NO NO NOD-Melezitose NO NO NO NO NO NO NO NO YES NO NO NO L-Sorbose NO NO NO NONO NO NO NO NO NO NO NO Citramalic acid NO NO NO NO NO NO NO NO NO NO NONO Oxalic acid NO NO NO NO NO NO NO NO NO NO NO NO L-Arginine NO NO NONO NO NO NO NO NO NO NO NO L-Valine YES YES NO YES YES NO NO NO NO NOYES NO γ-Cyclodextrin NO NO NO NO YES NO NO NO NO NO NO NO D-arabinoseNO NO NO NO NO NO NO YES NO NO NO NO Maltitol NO NO YES YES NO NO NO NONO NO NO YES Stachyose NO NO NO NO NO NO NO NO NO NO NO NO D-GlucosamineYES YES YES YES YES YES YES YES YES NO YES YES Oxalomalic acid YES YESYES YES NO YES NO NO YES NO YES YES Glycine NO NO NO NO NO NO NO NO NONO NO NO D,L-Carnitine YES YES NO NO NO NO NO NO NO NO NO NO Dextrin NONO NO YES YES NO NO YES YES YES NO NO D-arabitol NO NO NO NO NO NO NOYES NO NO NO NO a-Methyl-D- NO NO NO NO NO NO NO NO NO NO NO NOGlucoside D-Tagatose NO NO NO NO NO NO NO YES NO NO NO NO 2-Hydroxy NONO NO NO NO NO NO NO NO NO NO NO benzoic acid Quinic acid NO NO NO NO NONO NO NO NO NO NO NO L-Histidine NO NO NO NO NO YES NO NO NO YES NO NOSec-Butylamine NO NO NO NO NO NO NO NO NO NO NO NO Gelatin NO NO YES YESNO NO NO NO NO NO NO YES L-arabitol NO NO NO NO NO NO NO NO NO NO NO NOβ-Methyl-D- NO NO NO YES NO NO NO YES NO YES NO NO Galactoside TuranoseNO NO YES YES NO NO NO NO NO NO NO NO 4-Hydroxy NO NO NO NO NO NO NO NONO NO NO NO benzoic acid D-Ribono- NO NO NO NO NO NO NO NO NO NO NO NO1,4-Lactone L-Homoserine NO NO NO NO NO NO NO NO NO NO NO NOD,L-Octopamine YES YES YES YES YES NO NO NO YES NO YES YES Glycogen NONO NO NO NO NO NO YES NO NO NO NO Arbutin NO NO YES YES YES NO YES YESYES NO NO YES 3-Methyl Glucose NO NO NO NO NO NO NO YES NO NO NO NOXylitol NO NO NO YES NO NO NO NO NO NO NO NO β-Hydroxy NO NO NO NO NO NOYES YES NO NO NO NO butyric acid Sebacic acid NO NO NO NO NO NO NO NO NONO NO NO Hydroxy-L- NO NO NO NO NO NO YES NO NO NO NO NO ProlinePutrescine NO YES NO NO NO NO YES NO NO NO NO NO Inulin NO NO YES YESYES YES NO NO NO NO NO NO 2-Deoxy-D- NO NO NO NO NO NO NO YES NO YES NONO Ribose β-Methyl-D- NO NO NO NO NO NO NO NO NO NO NO NO Glucuronicacid N-Acetyl-D- NO NO NO NO NO NO NO NO NO NO NO NO glucosaminitolγ-Hydroxy NO NO NO NO NO NO NO NO NO NO NO NO butyric acid Sorbic acidNO NO NO NO NO NO NO NO NO NO NO NO L-Isoleucine YES NO NO NO NO NO NOYES NO NO YES NO Dihydroxy NO NO NO YES NO NO YES YES NO NO NO NOacetone Laminarin NO NO NO NO NO NO NO NO NO NO NO NO i-Erythritol NO NONO NO NO NO NO NO NO NO NO NO a-Methyl-D- NO NO NO NO NO NO NO NO NO NONO NO Mannoside γ-amino YES YES NO NO NO YES NO NO NO NO NO NO butyricacid a-Keto-valeric NO NO NO NO NO NO NO NO NO NO NO NO acid Succinamicacid NO NO NO NO NO NO NO NO NO NO NO NO L-Leucine YES NO NO NO NO NO NONO NO NO NO NO 2,3-Butanediol YES NO NO NO NO NO NO NO NO NO NO NOMannan NO NO NO NO NO NO NO NO NO NO NO NO D-Fucose NO NO NO NO NO NO NONO NO NO NO NO β-Methyl-D- NO NO NO NO NO NO NO NO NO NO NO NO Xylosided-amino NO NO NO NO NO NO NO NO NO NO NO NO valeric acid Itaconic acidNO NO NO NO NO NO YES YES NO NO NO NO D-Tartaric acid NO NO NO NO NO NONO NO NO NO NO NO L-Lysine NO NO NO NO NO NO NO NO NO NO NO NO2,3-Butanone NO NO NO NO NO NO NO NO NO NO NO NO Pectin NO NO NO NO NONO NO YES NO NO NO NO 3-0-β-D- NO NO NO NO NO NO NO NO NO NO NO NOGalactopyranosyl- D-arabinose Palatinose NO NO YES YES YES NO NO NO NONO NO YES Butyric acid NO NO NO NO NO NO NO NO NO NO NO NO 5-Keto-D- NOYES NO NO NO YES NO YES NO NO NO NO Gluconic acid L-Tartaric acid YESYES NO NO NO YES NO YES NO NO NO NO L-Methionine NO NO NO NO NO NO NO NONO NO NO NO 3-Hydroxy 2- NO NO NO NO NO NO NO NO NO NO NO NO Butanone

Twelve SYM strains of culturable bacteria belonging to OTUs present inlandrace and wild corn and wheat seeds that are present in lower levelsin modern corn and wheat seeds were tested for sole carbon substrateutilization using BIOLOG PM1 and PM2A MicroPlates. The most utilizedsubstrates by these strains are L-alanine, L-galactonic-acid-γ-lactone,maltose, maltotriose, D-cellobiose, gentiobiose, and D-glucosamine. Theleast utilized substrates by L-asparagine, L-glutamine, D-aspartic acid,tricarballylic acid, L-serine, L-fucose, 1,2-propanediol, D-threonine,L-threonine, succinic acid, fumaric acid, bromo succinic acid,D-L-a-glycerol phosphate, a-keto-butyric acid, a-hydroxy butyric acid,acetoacetic acid, glucuronamide, glycolic acid, mono methyl succinate,glyoxylic acid, phenylethyl-amine, and L-malic acid.

The substrates most utilized by a large number of the culturablebacteria belonging to core OTUs are mucic acid, L-arabinose,L-galactonic-acid-γ-lactone, N-acetyl-D-glucosamine, maltose,maltotriose, and D-cellobiose. These core bacteria did not utilizesedoheptulosan, oxalic acid, 2-hydroxy benzoic acid, quinic acid,mannan, L-methionine, N-acetyl-D-glucosaminitol, sorbic acid,2,3-butanone, succinic acid, phenylethyl-amine, and 3-hydroxy 2-butanoneas sole carbon sources. Results for the culturable fungi belonging tocore OTUs indicate that D-sorbitol, L-arabinose, N-acetyl-D-glucosamine,glycerol, tween 40, tween 80, D-gluconic acid, L-proline, a-D-glucose,D-trehalose, maltose, lactulose, D-mannose, D-mannitol, sucrose,D-cellobiose, L-glutamic acid, L-ornithine, and L-pyroglutamic acid arecarbon substrates that are utilized by a large number of the endophytestrains examined here. The carbon substrate that seemed to be notutilized by fungi in these assays is 2-deoxy-D-ribose. All othersubstrates could be utilized as a sole carbon nutrient by at least onefungi SYM strain.

Example 6 Testing of Culturable Bacterial and Fungal EndophytesBelonging to OTUs Present in Landrace and Wild Corn and Wheat Seeds thathave been Lost in Modern Corn and Wheat Seeds

The results shown above demonstrate that culturable microbes belongingto the same OTUs present in landrace and wild corn and wheat seeds thathave been lost in modern corn and wheat seeds possess activities thatcould impart beneficial traits to a plant upon colonization. The aim ofthe experiments in this section addresses the ability of theseculturable bacterial and fungal endophytes to confer beneficial traitson a host plant. Several different methods were used to ascertain this.First, plants inoculated with bacteria or fungi were tested underconditions without any stress to determine whether the microbe confersan increase in vigor. Second, endophyte-inoculated plants were testedunder water stress conditions to test whether the microbes confer anincrease in tolerance to this stress. These growth tests were performedusing growth assays on filter paper.

Seeds, Seed Sterilization and Seed Inoculation

Corn seeds were surface-sterilized with chlorine gas as described forExample 5. Inocula were also prepared and seeds inoculated as describedin Example 5.

Filter Paper Growth Assay

Bacterial endophytes isolated from seeds as described herein were testedfor their ability to promote plant growth under normal and stressedconditions by inoculating maize seeds with those endophytes andgerminating them on filter paper. Each bacterial endophyte to be testedwas streaked out onto 20% Tryptic Soy Agar, forming a lawn on regularPetri dishes (9 cm in diameter). Once the bacteria grew to high density,which happened after one or two days depending on the bacterial growthrate, a plate per bacterial strain was scraped with the aid of a sterileloop (filling the entire hole of the loop and producing a droplet ofbacterial biomass of about 20 mg). The bacteria collected in this waywere transferred into 1 ml of sterile 50 mM Phosphate Buffer Saline(PBS) in a microcentrifuge tube and fully resuspended by vortexing for˜20 sec at maximum speed. This method achieves highly concentrated(˜0.5-1 optical density, corresponding to about 10⁸ CFU/mL) and viablebacteria pre-adapted to live coating a surface.

Inoculation of seeds was performed by aliquoting ˜100 seeds into a 50 mlsterile test tube with conical bottom. Sodium Alginate (SA) was used asa sticker and added to the seeds in a proportion of 8.4 ml/kg of seed.After applying the appropriate volume of SA with the aid of an automatedpipette, the seeds were shook to ensure homogeneous coating. Immediatelyafter adding the SA, an equal volume of the bacterial suspension wasadded to the seeds and these were gently shooked to ensure homogeneouscoating.

Filter papers were autoclaved and placed into Petri dishes, and thenpresoaked with treatment solutions. To simulate normal conditions, 4 mLsterile water was added to the filters. Drought and saline stresses wereinduced by adding 4 mL 8% PEG 6000 solution or 100 mM NaCl to the filterpapers. Eight seeds were plated in triplicate for each condition tested.The Petri dishes were sealed with surgical tape to avoid evaporativewater loss and premature drying of the filter papers, randomized insidecardboard boxes to avoid position effects and placed in a growth chamberset at 22° C., 60% relative humidity, in the dark for five days.

Scoring of Results and Data Analysis

Once the seedlings had been growing for the prescribed period of time,they were removed from petri dishes, mounted on black cardboard backing,and photographed. After the seedlings were photographed, the images wereprocessed to recover phenotypic measurements for further statisticalanalysis. The image-processing pipeline consisted of a cropping methodthat isolated the seedling assay from the peripheral metadata, asegmentation method that isolated individual seedlings from thebackground, and a rapid phenotyping tool that measured features ofisolated seedling root morphologies.

Raw images were cropped using matrix rotation and subsampling methodsincluded in the numpy python package. (van Rossum 2006, van der Walt2011) Further segmentation on the cropped images was performeddifferently depending on crop genotype: soy seedlings were segmentedusing a watershed algorithm on the discreet Sobel gradient of thegrayscale cropped image, while wheat and corn seedlings were segmentedusing Otsu's binary thresholding algorithm on the discreet Laplaciangradient of a Gaussian kernel convolved with the greyscale cropped image(Ando 2000, Otsu 1979). Image processing was performed using toolsincluded in the Scikit-Image python package (vanderWalt, et al., 2014).Finally, the whole seedling biomass (root and shoot) of each treatmentwas determined using our own image processing metric.

Experimental Results

The effects of bacterial and fungal endophytes belonging to OTUs presentin landrace and wild seeds, and combinations of bacterial endophytes orfungal endophytes, on the growth of corn seeds in a filter paper assayis shown in Table 34A and 34B.

TABLE 34A Growth of corn seeds treated with bacterial endophytesbelonging to OTUs present in landrace and wild seeds that are found atlower levels in modern seeds. Biological Water Biological SaltBiological Strain SEQ ID NO. Normal Effect? stress Effect? stressEffect? SYM00013 3590 1 no 2 yes 1 yes SYM00018 3592 1 no 0 no 2 yesSYM00021b 3594 1 no 1 yes 0 yes SYM00025 3595 2 yes 0 no 1 yes SYM000433598 0 yes 0 yes 0 yes SYM00044 3599 1 no 0 yes 0 yes SYM00074 3608 1 no1 yes 2 yes SYM00184 3621 1 yes 2 yes 0 no SYM00219 3624 0 yes 0 yes 0yes SYM00545 3637 0 yes 0 yes 0 yes SYM00549 3638 0 yes 0 yes 0 yesSYM00617 3645 1 no 2 yes 0 yes SYM00620 3646 1 yes 2 yes 0 yes SYM006463651 0 yes 0 yes 0 yes SYM00662 3653 1 yes 0 yes 1 no SYM00905 3663 1yes 0 no 1 yes 0 indicates <0% effect, 1 indicates <20% effect, 2indicates <40% effect, 3 indicates >40% effect. For Biological Effect:yes indicates >5% or <−5% effect, no indicates effect between −5% and+5%.

In the salt stress, 12.5% of the microbial inoculants elicited >40%improvement on plant phenotype compared to seeds that were treated withthe formulation suggesting a role in improving plant vigor under a saltstress condition. One of the two top performers is Pantoea sp.(SYM00018) and these strains were among the highest auxin producerstested which may indicate important beneficial traits for the plantassociated with this genus. Under water stress, 25% of the microbialinoculants elicited >40% improvement on plant phenotype suggesting arole in improving plant vigor under a water stress condition. The fourstrains which provided the largest improvement to plant phenotype camefrom different genera, and two of them, SYM00013 and SYM00184, showedthe ability to utilize a number of different carbon substratesincluding: L-Arabinose, N-Acetyl-D-Glucosamine, L-Alanine, D-Galactose,a-Hydroxy Glutaric acid-?-lactone, L-Alanyl-Glycine, D-Xylose, Mucicacid, D-Alanine and Uridine. This suggests that they may have similarroles in providing water stress protection for the plant. Under normalcondition, 6.25% of the microbial inoculants elicited >40% improvementon plant phenotype compared to seeds that were treated with theformulation suggesting a role in improving plant vigor under a saltstress condition. The top performer under normal condition is Pantoeasp. (SYM00018) and these strains were among the highest auxin producerstested which may indicate important beneficial traits for the plantassociated with this genus.

TABLE 34B Growth of corn seeds treated with combinations of bacterialendophytes belonging to OTUs present in landrace and wild seeds that arefound at lower levels in modern seeds. SEQ SEQ Water Strain 1 ID NO.Strain 2 ID NO. Normal * stress * SYM00025 3595 SYM00044 3599 0 0/aSYM00043 3598 SYM00018 3592 − 0 SYM00074 3608 SYM00184 3621 0 − SYM005493638 SYM00617 3645 0/−b 0/a SYM00646 3651 SYM00662 3653   +/a, b, cSYM00662 3653 SYM00025 3595 −/−b +/a SYM00905 3663 SYM00043 3598 −/−b0 * Any symbol to the left of the “/” pertains to primary radicle lengthwith +, 0, − denoting an increase, no change, or decrease relative tocontrol seedling radicles, respectively. The scale (a-e) to the right ofthe “/” pertains to relative increases or decreases in secondarycharacteristics of the seedlings as follows: a) root hair development,b) lateral root number, c) lateral root size, d) shoot length, and e)root thickness.

A beneficial plant microbiome is likely made up of multiple strains thatoccupy stress protection niches within the plant. These particularbacterial endophyte combinations were evaluated in a germination assaysto test the improvement on the plant phenotype conferred by inoculationwith multiple bacterial strains. In water stressed plants thecombination SYM00646/SYM00662 and SYM00662/SYM00025 provided improvementin plant phenotype compared to the formulation control.

Example 8 Identification and Characterization of Culturable Bacterialand Fungal Endophytes Belonging to Core OTUs Isolation andIdentification of Culturable Microbes

In order to better understand the role played by core seed-derivedendophytic microbes in improving the vigor, general health and stressresilience of agricultural plants, we identified culturable microbesthat belong to the same OTUs as the core OTUs of Tables 13 and 14. Usingthe same methods as in Example 3 and other techniques known in the art,bacterial and fungal endophytes were cultured were from a variety ofplant parts and a variety of plants. To accurately characterize theisolated microbial endophytes, colonies were submitted for marker genesequencing, and the sequences were analyzed to provide taxonomicclassifications. Among the cultured microbes, those with at least 97%16S or ITS sequence similarity to certain microbes of Tables 13 and 14were identified. Those microbes are listed in Tables 35A and 35B.

TABLE 35A Cultured bacterial isolates belonging to the same OTUs ascertain bacteria of Table 13. Cultured SEQ ID NO SEQ ID NO Old OTU NewOTU bacterial of cultured of core of core of core isolate bacterialisolate bacterial taxa bacterial taxa bacterial taxa SYM00003 3588 20 58B0.9|GG99|238752 SYM00009 3589 20 58 B0.9|GG99|238752 SYM00013 3590 33 7B0.9|GG99|4327501 SYM00017A 3591 803 54 B0.9|GG99|5409 SYM00018 3592 313489 B0.9|GG97|2582263 SYM00020 3593 32 1255 B0.9|GG97|2582263 SYM000333596 1953 2912 B0.9|GG97|253061 SYM00050 3600 26, 28, 24, 25, OTU_3592,B0.9|GG97|816702, 1939, 1548 OTU_1384, B0.9|GG99|218527, OTU_3629,B0.9|GG97|639627, OTU_2970, B0.9|GG97|253061, OTU_3153,B0.9|GG97|4374146, OTU_115 B0.9|GG99|625742 SYM00053 3601 25, 27, 28,29, 2970, X, 1384, 32, 1953 X, X, 2912 SYM00062C 3603 15 62B0.9|GG99|685917 SYM00065 3604 11, 891, 892, 23, 3209, 3351,B0.9|GG99|2929397, 895 568 B0.9|GG99|4450360, B0.9|GG97|158370,B0.9|GG99|2185530 SYM00068 3606 33 7 B0.9|GG99|4327501 SYM00070 3607 302 B0.9|GG99|9943 SYM00103 3609 20 58 B0.9|GG99|238752 SYM00170 3619 102310 B0.9|GG99|1082594 SYM00183 3620 37 83 B0.9|GG99|4102407 SYM00184 362137 83 B0.9|GG99|4102407 SYM00207 3622 12 131 B0.9|GG99|4298641 SYM002123623 18 38 B0.9|GG99|29974 SYM00219 3624 18, 981, 988 38, 3473, 106B0.9|GG99|29974, B0.9|GG99|156425, B0.9|GG99|277294 SYM00234 3625 102310 B0.9|GG99|1082594 SYM00236 3626 9 69 B0.9|GG99|175931 SYM00248 362730 2 B0.9|GG99|9943 SYM00249 3628 12, 1047 131, 212 B0.9|GG99|4298641,B0.9|GG99|14492 SYM00507 3630 12, 1047 131, 212 B0.9|GG99|4298641,B0.9|GG99|14492 SYM00508 3631 25 2970 B0.9|GG97|253061 SYM00525 3632 328 B0.9|GG99|813062 SYM00538A 3633 891, 892  3209, 3351B0.9|GG99|4450360, B0.9|GG97|158370 SYM00538B 3634 1023 10B0.9|GG99|1082594 SYM00538i 3635 22 60 B0.9|GG99|105406 SYM00543 3636 1459 B0.9|GG99|144390 SYM00563 3639 988 106 B0.9|GG99|277294 SYM00617 3645988 106 B0.9|GG99|277294 SYM00620 3646 26, 28, 24, 25, OTU_3592,B0.9|GG97|816702, 1939, 1548 OTU_1384, B0.9|GG99|218527, OTU_3629,B0.9|GG97|639627, OTU_2970, B0.9|GG97|253061, OTU_3153,B0.9|GG97|4374146, OTU_115 B0.9|GG99|625742 SYM00627 3648 27 35B0.9|GG99|370327 SYM00628 3649 25, 27, 28, 29, 30, 1953 SYM00650 3652 337 B0.9|GG99|4327501 SYM00714 3656 803 54 B0.9|GG99|5409 SYM00905 3663 3783 B0.9|GG99|4102407 SYM00924 3664 9 69 B0.9|GG99|175931 SYM00963 366529 319 B0.9|GG99|295383 SYM00978 3668 29, 30, 31, 32, 1953 SYM00982 366622 60 B0.9|GG99|105406 SYM00987 3667 29 319 B0.9|GG99|295383 SYM009913669 1139, 81, 64 B0.9|GG99|988067, 1164 B0.9|GG99|4426695 SYM00999 36709 69 B0.9|GG99|175931 SYM01049 3671 25, 27, 28, 30, 1953

TABLE 35B Cultured fungal isolates belonging to the same OTUs as certainfungi of Table 14. SEQ ID NO Old OTU NEW OTU SEQ of core of core of coreStrain ID NO. fungal taxa fungal taxa fungal taxa SYM00034 3597 2965 7F0.9|UDYN|210204 SYM00061A 3602 2965 7 F0.9|UDYN|210204 SYM00066 36052965 7 F0.9|UDYN|210204 SYM00120 3610 2699 2 F0.9|UDYN|206476 SYM001223611 2701 45  F0.9|U97|025461 SYM00123 3612 2701 45  F0.9|U97|025461SYM00124 3613 2968 8 F0.9|UDYN|220700 SYM00129 3614 2698 1F0.9|UDYN|424875 SYM00135 3615 2698 1 F0.9|UDYN|424875 SYM00136 36162698 1 F0.9|UDYN|424875 SYM00151 3617 2698 1 F0.9|UDYN|424875 SYM001543618 2966, 2983, 3, 351, 728, 766 F0.9|UDYN|215392; 2987, 3002F0.9|U97|020374; F0.9|SF0|A8L3R2113:21340:17509;F0.9|SF0|A8L3R2102:11106:24468 SYM00566B 3640 2965 7 F0.9|UDYN|210204SYM00577 3642 2965 7 F0.9|UDYN|210204 SYM00590 3643 2965 7F0.9|UDYN|210204 SYM00603 3644 2965 7 F0.9|UDYN|210204 SYM00622 36472965 7 F0.9|UDYN|210204 SYM00629 3650 2965 7 F0.9|UDYN|210204 SYM006633654 2699 2 F0.9|UDYN|206476 SYM00696 3655 2699 2 F0.9|UDYN|206476SYM00741a 3657 2741 10  F0.9|UDYN|186595 SYM00741b 3658 2733, 2751, 5,883, 974 F0.9|UDYN|212600; 2799 F0.9|SF0|A8L3R1114:18309:4041; SYM007933659 2698 1 F0.9|UDYN|424875 SYM00795 3660 2965 7 F0.9|UDYN|210204SYM00854 3661 2741 10 F0.9|UDYN|186595 SYM00880 3662 2699 2F0.9|UDYN|206476 SYM01300 3672 2965 7 F0.9|UDYN|210204 SYM01303 36732968 8 F0.9|UDYN|220700 SYM01310 3674 2698 1 F0.9|UDYN|424875 SYM013113675 2698 1 F0.9|UDYN|424875 SYM01314 3676 2965 7 F0.9|UDYN|210204SYM01315 3677 2733, 2751, 5, 883, 974 F0.9|UDYN|212600; 2799F0.9|SF0|A8L3R1114:18309:4041; SYM01325 3678 2699 2 F0.9|UDYN|206476SYM01326 3679 2699 2 F0.9|UDYN|206476 SYM01327 3680 2733, 2751, 5, 883,974 F0.9|UDYN|212600; 2799 F0.9|SF0|A8L3R1114:18309:4041; SYM01328 36812699 2 F0.9|UDYN|206476 SYM01333 3682 2737 4 F0.9|SF97|43 SYM15811 36832699 2 F0.9|UDYN|206476 SYM15820 3684 2980 50  F0.9|UDYN|177637 SYM158213685 2980 50  F0.9|UDYN|177637 SYM15825 3686 2966, 2983, 3, 351, 728,766 F0.9|UDYN|215392; 2987, 3002 F0.9|U97|020374;F0.9|SF0|A8L3R2113:21340:17509; F0.9|SF0|A8L3R2102:11106:24468 SYM158283687 2966, 2983, 3, 351, 728, 766 F0.9|UDYN|215392; 2987, 3002F0.9|U97|020374; F0.9|SF0|A8L3R2113:21340:17509;F0.9|SF0|A8L3R2102:11106:24468 SYM15831 3688 2698 1 F0.9|UDYN|424875SYM15837 3689 2966, 2983, 3, 351, 728, 766 F0.9|UDYN|215392; 2987, 3002F0.9|U97|020374; F0.9|SF0|A8L3R2113:21340:17509;F0.9|SF0|A8L3R2102:11106:24468 SYM15839 3690 2966, 2983, 3, 351, 728,766 F0.9|UDYN|215392; 2987, 3002 F0.9|U97|020374;F0.9|SF0|A8L3R2113:21340:17509; F0.9|SF0|A8L3R2102:11106:24468 SYM158473691 2698 1 F0.9|UDYN|424875 SYM15870 3692 2966, 2983, 3, 351, 728, 766F0.9|UDYN|215392; 2987, 3002 F0.9|U97|020374;F0.9|SF0|A8L3R2113:21340:17509; F0.9|SF0|A8L3R2102:11106:24468 SYM158723693 2966, 2983 3, 351 F0.9|UDYN|215392; F0.9|U97|020374 SYM15890 36942733, 2751, 5, 883, 974 F0.9|UDYN|212600; 2799F0.9|SF0|A8L3R1114:18309:4041; SYM15901 3695 2966, 2983, 3, 351, 728,766 F0.9|UDYN|215392; 2987, 3002 F0.9|U97|020374;F0.9|SF0|A8L3R2113:21340:17509; F0.9|SF0|A8L3R2102:11106:24468 SYM159203696 2966, 2983, 3, 351, 728, 766 F0.9|UDYN|215392; 2987, 3002F0.9|U97|020374; F0.9|SF0|A8L3R2113:21340:17509;F0.9|SF0|A8L3R2102:11106:24468 SYM15926 3697 2699 2 F0.9|UDYN|206476SYM15928 3698 2699 2 F0.9|UDYN|206476 SYM15932 3699 2966, 2983, 3, 351,728, 766 F0.9|UDYN|215392; 2987, 3002 F0.9|U97|020374;F0.9|SF0|A8L3R2113:21340:17509; F0.9|SF0|A8L3R2102:11106:24468 SYM159393700 2966, 2983, 3, 351, 728, 766 F0.9|UDYN|215392; 2987, 3002F0.9|U97|020374; F0.9|SF0|A8L3R2113:21340:17509;F0.9|SF0|A8L3R2102:11106:24468

Characterization of Culturable Microbes: Auxin, Acetoin and SiderophoreProduction

The culturable microbes belonging to the same OTUs as certain microbesof Tables 13 or Table 14 were then seeded onto 96 well plants and testedfor auxin, acetoin and siderophore production, using the methodsdescribed in Example 4. The results are presented in Tables 36A and 36B.

TABLE 36A Auxin, siderophore, and acetoin production by culturablebacteria belonging to core OTUs SEQ ID Secretes Produces Produces StrainNO. siderophores Auxin/Indoles Acetoin SYM00003 3588 2 1 0 SYM00009 35891 1 0 SYM00013 3590 0 1 0 SYM00017A 3591 1 3 0 SYM00018 3592 0 3 2SYM00020 3593 0 2 2 SYM00021b 3594 0 2 3 SYM00025 3595 1 3 2 SYM000433598 1 3 2 SYM00044 3599 1 1 3 SYM00050 3600 1 2 3 SYM00053 3601 1 1 2SYM00062C 3603 1 2 1 SYM00068 3606 2 2 0 SYM00070 3607 2 2 0 SYM000743608 2 3 0 SYM00103 3609 2 2 2 SYM00183 3620 0 2 1 SYM00184 3621 0 2 0SYM00207 3622 1 2 2 SYM00212 3623 2 2 3 SYM00219 3624 3 2 3 SYM002343625 2 2 2 SYM00236 3626 0 2 0 SYM00248 3627 1 2 0 SYM00249 3628 2 2 2SYM00506c 3629 0 2 2 SYM00507 3630 1 2 2 SYM00508 3631 0 3 2 SYM005253632 2 2 3 SYM00538A 3633 3 2 3 SYM00538B 3634 2 2 2 SYM00538i 3635 0 10 SYM00543 3636 0 3 1 SYM00545 3637 2 2 2 SYM00549 3638 2 2 2 SYM005633639 2 2 1 SYM00574 3641 3 1 0 SYM00617 3645 1 3 1 SYM00620 3646 1 3 0SYM00627 3648 0 1 3 SYM00628 3649 2 2 3 SYM00646 3651 3 2 3 SYM006503652 2 2 0 SYM00662 3653 1 1 1 SYM00714 3656 1 2 2 SYM00905 3663 3 2 2SYM00924 3664 2 2 2 SYM00963 3665 2 2 1 SYM00978 3668 2 2 1 SYM009823666 0 2 3 SYM00987 3667 1 3 2 SYM00991 3669 1 2 2 SYM00999 3670 1 1 3SYM01049 3671 1 1 0

In total, a very large proportion of the bacteria strains, 44 out of 55strains (80% of total) tested, were able to utilize Tryptophansupplemented in the medium and showed a detectable level of pink or redcolor development (the diagnostic feature of the assay suggesting auxinor indolic compound production). These include 8 Bacillus spp., 5Paenibacillus spp., 6 Pantoea spp., and 5 Enterobacter spp. 10 strains(18% of total) had particularly strong production of auxin or indolecompounds. As for acetoin production, 32 out of 21 strains (58% oftotal) tested showed a detectable level of pink or red color (a proxy ofacetoin production). These include 6 Enterobacter spp., 5 Paenibacillusspp., and 4 Bacillus spp. Particularly, 12 of these 32 strains hadstrong production of acetoin. 23 out of 55 strains (42% of total) testedshowed a detectable level of siderophore accumulation. These include 4Bacillus spp., 4 Paenibacillus spp., 4 Enterobacter spp., and 3Pseudomonas spp. Among these 23 strains, 5 strains showed very strongaccumulation of siderophore.

TABLE 36B Auxin, siderophore, and acetoin production by culturable fungibelonging to core OTUs SEQ ID Secretes Produces Produces Strain NO.siderophores Auxin/Indoles Acetoin SYM00034 3597 1 0 0 SYM00061A 3602 10 2 SYM00066 3605 1 0 0 SYM00120 3610 1 0 0 SYM00122 3611 0 0 0 SYM001233612 1 0 3 SYM00124 3613 1 1 0 SYM00129 3614 0 1 0 SYM00135 3615 0 1 0SYM00136 3616 0 0 1 SYM00151 3617 1 1 0 SYM00154 3618 0 0 0 SYM00566B3640 3 0 0 SYM00577 3642 0 0 1 SYM00590 3643 0 1 2 SYM00603 3644 2 1 0SYM00622 3647 1 0 2 SYM00629 3650 0 1 2 SYM00663 3654 2 1 2 SYM006963655 2 0 0 SYM00741b 3658 0 0 0 SYM00793 3659 1 0 0 SYM00795 3660 1 0 1SYM00854 3661 2 0 2 SYM00880 3662 2 1 2 SYM01300 3672 2 1 0 SYM013033673 — — — SYM01310 3674 0 2 0 SYM01311 3675 0 0 0 SYM01314 3676 2 1 0SYM01315 3677 0 0 0 SYM01325 3678 0 0 2 SYM01326 3679 0 0 2 SYM013273680 2 1 2 SYM01328 3681 1 0 0 SYM01333 3682 0 0 0 SYM15811 3683 3 1 0SYM15820 3684 1 0 0 SYM15821 3685 1 0 0 SYM15825 3686 0 0 2 SYM158283687 0 0 2 SYM15831 3688 2 1 2 SYM15837 3689 1 0 0 SYM15839 3690 2 0 0SYM15847 3691 0 0 0 SYM15870 3692 0 0 0 SYM15872 3693 0 0 1 SYM158903694 0 0 2 SYM15901 3695 0 0 2 SYM15920 3696 2 0 2 SYM15926 3697 1 2 0SYM15928 3698 0 0 0 SYM15932 3699 0 0 0 SYM15939 3700 0 1 0

In total, most fungi were not able to utilize L-Tryptophan supplementedin the medium. 17 out of 51 strains tested (31% of total) showed adetectable level of pink or red color development (the diagnosticfeature of the assay suggesting auxin or indolic compound production).These include 5 Acremonium spp., 4 Alternaria spp., and 3 Fusariam spp.Only 2 strains (4% of total) had particularly strong production of auxinor indole compounds. As for acetoin production, 17 out of 54 strains(31% of total) tested showed a detectable level of pink or red color (aproxy of acetoin production). These include 5 Fusarium spp., 4Alternaria spp., and 4 Acremonim spp. Particularly, only 1 of these 17strains had strong production of acetoin. 13 out of 21 strains (24% oftotal) tested showed a detectable level of siderophore accumulation.These include 4 Alternaria spp., 4 Acremonium spp., and 3 Fusarium spp.Among these 13 strains, 2 strains showed very strong accumulation ofsiderophore.

Characterization of Culturable Microbes: Substrate Use

The BIOLOG protocol was conducted in the same manner as describedpreviously.

The ability of a strain to utilize a specific carbon substrate in theBIOLOG PM1 or PM2A MicroPlates could be determined by colorimetric assayand increased turbidity due to cell growth in that particular well(Tables 36C, 36D, 36E and 36F).

TABLE 36C Substrate utilization as determined by BIOLOG PM1 MicroPlatesby culturable bacteria belonging to core OTUs. Strain SYM SYM SYM SYMSYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM Substrate 103 1049 1317A 18 183 184 20 207 212 219 234 236 248 249 260 D-Serine NO NO NO NONO NO NO NO NO NO NO NO NO YES NO NO D-Glucose- NO NO NO YES NO NO NO NONO YES NO NO NO YES YES NO 6-Phosphate L-Asparagine NO NO NO YES NO NONO NO NO NO NO NO NO NO YES YES L-glutamine NO NO NO NO NO NO NO NO NONO NO NO NO NO YES NO Glycyl-L- NO YES YES NO NO NO NO NO NO YES NO NONO NO NO NO Aspartic acid Glycyl-L- YES NO NO NO NO YES YES NO YES YESNO NO NO NO NO YES Glutamic acid Glycyl-L- NO NO NO NO NO YES YES NO NONO NO NO NO NO NO NO Proline L-Arabinose NO YES YES YES YES NO YES YESNO YES NO YES YES YES YES YES D-Sorbitol NO NO NO YES NO NO YES NO NO NONO NO NO NO NO NO D-Galactonic NO YES YES NO YES NO NO YES NO NO YES NONO NO NO NO acid-?-lactone D-Aspartic acid NO NO NO NO NO NO NO NO NOYES NO NO NO NO NO NO m-Tartaric acid NO YES YES NO YES NO NO YES NO YESNO NO NO NO NO NO Citric acid NO NO NO NO NO NO NO NO NO NO NO NO NO NOYES YES Tricarballylic NO NO NO NO NO NO NO NO NO YES NO NO NO NO NO NOacid p-Hydroxy NO NO NO YES NO NO NO NO NO NO NO NO NO NO NO NO Phenylacetic acid N-Acetyl-D- NO NO YES YES YES YES YES YES YES YES YES YES NOYES YES NO Glucosamine Glycerol NO NO NO YES NO NO NO NO NO YES NO YESNO YES NO YES D-L-Malic acid NO NO NO YES NO NO YES NO YES YES NO YES NONO NO YES D-Glucosaminic NO NO NO YES YES NO NO YES NO NO NO NO NO NO NONO acid D-Glucose-1- NO NO NO YES YES NO NO YES NO YES NO NO NO NO YESNO Phosphate m-Inositol NO NO NO YES YES NO YES YES NO YES NO NO NO YESYES YES L-Serine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO YESm-Hydroxy NO NO NO NO NO NO NO NO YES NO NO NO NO NO NO NO Phenyl Aceticacid D-Saccharic NO NO NO YES NO NO YES NO NO YES NO NO NO NO NO YESacid L-Fucose NO NO NO YES NO NO NO NO NO YES NO NO NO NO NO NO D-RiboseNO YES NO YES YES YES YES NO NO YES NO NO NO YES YES NO 1,2-PropanediolNO NO NO NO NO NO NO NO NO YES NO NO NO NO NO YES D-Fructose- NO NO NOYES YES NO NO NO NO YES NO NO NO NO YES NO 6-Phosphate D-Threonine NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO L-Threonine NO NO NO NO NO NONO NO YES NO NO NO NO NO NO YES Tyramine NO NO YES YES YES YES NO YESYES YES YES YES NO YES YES NO Succinic acid NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO D-Glucuronic NO NO NO NO NO NO NO NO NO NO NO NO NONO YES NO acid Tween 20 NO NO NO NO NO NO YES NO NO NO NO NO NO NO NOYES Tween 40 NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO YES Tween 80YES YES NO NO NO YES YES NO NO NO NO NO NO NO NO YES Fumaric acid NO NONO NO NO NO NO NO NO YES NO NO NO NO NO YES L-Alanine YES NO YES YES YESYES YES YES YES YES YES YES YES YES NO YES D-Psicose NO NO NO YES YES NONO YES NO NO NO NO NO NO NO NO D-Galactose NO YES YES YES YES NO YES YESNO NO YES NO NO YES YES NO D-Gluconic YES NO NO NO YES NO NO NO NO YESNO YES NO NO NO YES acid L-Rhamnose NO NO NO YES YES NO NO YES NO YESYES YES NO YES YES NO a-Keto-Glutaric YES NO NO YES NO YES NO NO YES YESNO NO YES NO NO YES acid a-Hydroxy NO NO YES YES NO NO YES NO NO YES NONO YES NO NO YES Glutaric acid-?- lactone Bromo succinic NO NO NO NO NONO NO NO NO NO NO NO NO NO NO YES acid L-Alanyl- YES NO YES YES YES YESYES YES YES YES NO YES NO YES NO YES Glycine L-Lyxose NO NO NO NO YES NONO YES NO NO NO NO NO YES NO NO L-Aspartic acid YES NO NO NO NO YES NONO YES NO NO NO YES YES YES YES D-L-a-Glycerol NO NO NO NO NO NO NO NONO NO NO NO NO NO NO YES phosphate D-Fructose NO NO NO YES NO NO YES YESNO YES NO YES NO YES NO NO a-Keto-Butyric NO NO NO NO NO NO NO NO YESYES NO NO NO NO NO NO acid a-Hydroxy NO NO NO NO NO NO NO NO NO YES NONO NO NO NO YES Butyric acid Propionic acid NO NO NO YES YES NO NO NOYES YES NO NO NO NO NO YES Acetoacetic NO NO NO NO NO NO NO NO NO NO NONO NO NO NO YES acid Glucuronamide NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO L-Proline NO NO NO NO YES YES NO NO YES YES NO YES YES YESNO YES D-Xylose YES YES YES YES YES YES YES YES NO YES NO YES YES YES NOYES Acetic acid NO NO NO YES YES NO YES NO YES YES NO YES YES NO NO YESa-Methyl- NO NO NO YES NO NO NO NO NO YES YES YES NO YES NO NOD-Galactoside β-Methyl-D- NO NO NO YES YES NO YES YES NO YES YES YES NOYES YES YES glucoside Mucic acid YES YES YES YES YES YES NO YES YES YESNO YES NO YES NO YES N-acetyl-β-D- NO NO NO NO NO NO NO NO NO NO NO YESNO NO NO YES Mannosamine Pyruvic acid NO YES NO YES YES YES YES YES YESYES YES YES NO NO YES YES D-Alanine YES NO YES YES YES YES YES YES NO NOYES NO NO NO YES NO L-Lactic acid NO NO NO NO NO NO NO NO NO NO NO NO NONO NO YES a-D-Glucose NO YES NO YES YES YES YES NO NO YES NO YES NO YESNO NO a-D-Lactose NO NO NO NO NO YES YES NO NO YES NO YES NO YES NO NOAdonitol NO NO NO YES YES YES NO NO YES NO NO YES NO NO NO NO Glycolicacid YES NO NO NO NO NO NO NO YES YES NO NO NO NO NO YES Mono Methyl NOYES NO NO NO NO NO NO YES NO NO YES NO NO NO YES Succinate L-Galactonic-YES YES NO YES YES YES YES YES YES YES YES YES NO NO NO YESacid-?-lactone D-Trehalose YES NO NO YES NO NO NO NO NO NO NO YES NO YESNO NO Formic acid NO NO NO NO YES NO NO YES NO NO NO NO NO YES NO YESMaltose NO YES NO YES YES YES YES YES NO YES YES YES NO YES YES YESLactulose NO NO NO YES NO YES YES NO NO YES NO YES NO NO NO NOMaltotriose NO NO NO YES YES YES YES YES NO YES YES YES NO YES YES YESGlyoxylic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO YES MethylNO NO NO NO NO NO NO NO NO YES NO YES NO NO NO YES PyruvateD-Galacturonic YES NO NO YES NO YES NO NO YES NO NO NO NO NO NO YES acidD-Mannose NO YES NO YES YES YES YES NO NO NO NO YES NO YES NO NOD-Mannitol NO NO NO YES YES NO YES NO NO NO NO YES NO YES YES YESD-Melibiose NO NO NO YES YES YES YES NO NO YES YES YES NO YES NO NOSucrose NO NO NO YES NO YES YES NO NO NO NO YES NO NO YES NO 2-Deoxy NONO NO NO YES NO NO NO NO NO NO NO NO YES NO YES adenosine D-CellobioseNO YES NO YES YES YES YES YES YES YES YES YES NO YES YES YES D-Malicacid NO YES NO NO NO NO NO NO NO NO NO NO NO YES NO YES Phenylethyl- NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO amine Dulcitol NO NO NO NONO NO NO NO NO NO YES NO NO NO NO NO L-Glutamic NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO acid Thymidine NO NO NO NO YES NO NO NO YES YESNO YES NO NO YES YES Uridine YES NO YES YES YES YES YES NO YES YES NOYES NO NO YES YES Adenosine YES NO NO YES YES NO NO NO NO YES YES YES NOYES NO YES Inosine NO NO NO NO NO NO YES NO YES NO NO YES NO NO NO NOL-Malic acid NO NO NO NO NO NO NO NO YES NO NO NO NO NO NO YES2-Aminoethanol YES NO NO YES YES YES YES NO NO NO NO YES NO NO NO YESStrain SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYMSYM Substrate 290 292 3 43 50 5066 508 525 53 538A 538B 538i 543 563 57457B 617 D-Serine NO YES NO NO YES NO NO NO YES NO NO NO YES NO NO NO NOD-Glucose- NO NO NO YES YES NO YES NO YES NO NO NO YES NO NO NO NO6-Phosphate L-Asparagine NO NO NO NO NO NO NO NO NO NO NO NO YES NO NONO NO L-glutamine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOGlycyl-L- NO NO NO YES YES NO NO NO YES NO NO NO NO NO NO NO NO Asparticacid Glycyl-L- NO NO NO NO NO NO NO NO NO NO NO NO YES NO YES NO NOGlutamic acid Glycyl-L- NO NO NO NO YES NO NO NO YES NO NO NO NO NO NOYES NO Proline L-Arabinose YES YES YES YES YES NO NO NO YES NO YES YESYES YES YES YES NO D-Sorbitol NO NO NO NO YES NO NO YES YES NO NO NO NONO NO NO NO D-Galactonic NO NO NO YES NO NO NO NO NO NO NO NO NO NO NONO NO acid-?-lactone D-Aspartic acid NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO m-Tartaric acid NO NO NO YES NO NO NO NO NO NO NO NO NONO NO NO NO Citric acid NO YES NO NO NO NO NO NO NO NO NO YES YES NO YESNO NO Tricarballylic NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOacid p-Hydroxy NO NO NO NO YES NO NO NO NO NO NO NO NO NO NO NO NOPhenyl acetic acid N-Acetyl-D- YES YES YES YES YES NO NO YES YES YES YESYES YES NO YES NO NO Glucosamine Glycerol YES YES NO YES YES NO NO NO NONO YES NO YES NO YES NO NO D-L-Malic acid YES YES NO YES NO NO YES YESNO YES YES YES YES NO YES NO YES D-Glucosaminic NO NO YES YES NO NO YESNO NO NO NO NO NO NO NO NO NO acid D-Glucose-1- NO NO NO YES YES NO YESNO NO NO NO NO NO NO NO NO NO Phosphate m-Inositol NO YES NO YES YES NONO YES YES NO YES YES YES NO YES NO NO L-Serine NO NO NO NO NO NO NO NONO NO NO NO YES NO YES NO NO m-Hydroxy NO NO NO NO YES NO NO NO YES NONO NO NO NO NO YES NO Phenyl Acetic acid D-Saccharic NO YES NO YES YESNO YES NO NO NO NO YES YES NO YES NO NO acid L-Fucose YES NO NO NO NO NONO NO NO NO NO NO YES NO NO YES NO D-Ribose YES YES YES YES NO NO NO YESNO NO NO NO YES NO NO NO NO 1,2-Propanediol YES NO NO NO NO NO NO NO NONO NO NO NO NO YES NO NO D-Fructose- NO NO NO NO YES NO YES NO YES NO NONO NO NO NO NO NO 6-Phosphate D-Threonine YES NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO L-Threonine YES NO NO NO NO NO NO NO NO NO NO NOYES NO YES NO NO Tyramine NO YES YES NO NO NO NO NO NO NO NO NO YES NONO NO NO Succinic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO D-Glucuronic NO NO NO NO YES NO NO NO NO NO NO NO NO NO NO NO NO acidTween 20 YES NO NO NO NO NO NO NO NO NO YES NO NO NO YES YES NO Tween 40YES NO NO YES NO NO NO NO NO NO NO NO NO NO YES NO NO Tween 80 YES NO NONO NO NO NO NO NO NO NO NO YES NO YES NO NO Fumaric acid YES YES NO NONO NO NO NO NO NO NO NO NO NO YES NO NO L-Alanine YES YES YES YES YES NONO YES YES NO NO NO YES NO YES NO NO D-Psicose NO NO NO YES NO NO NO NONO NO NO NO NO NO NO NO NO D-Galactose YES YES NO YES YES NO YES YES NONO YES NO NO NO NO YES NO D-Gluconic YES YES NO YES YES NO YES NO YES NOYES NO YES NO YES NO NO acid L-Rhamnose YES YES NO YES YES NO YES YESYES NO YES NO NO YES NO YES YES a-Keto-Glutaric NO YES NO NO YES NO NONO YES YES YES YES YES NO YES NO NO acid a-Hydroxy NO NO NO NO YES NO NONO NO NO NO YES NO NO YES NO NO Glutaric acid- ?-lactone Bromo succinicNO YES NO NO NO NO NO NO NO NO NO NO YES NO YES NO NO acid L-Alanyl- YESYES YES NO YES NO NO YES NO YES YES YES YES NO YES NO NO GlycineL-Lyxose NO NO NO YES YES NO YES NO NO NO NO NO NO NO NO NO NOL-Aspartic acid NO YES NO NO YES NO YES YES NO NO YES YES YES NO YES NONO D-L-a-Glycerol NO NO NO NO NO NO NO NO NO NO NO NO NO NO YES NO NOphosphate D-Fructose YES YES NO YES YES NO NO YES YES NO YES NO YES NONO NO NO a-Keto-Butyric NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO acid a-Hydroxy YES NO NO NO NO NO NO NO NO NO NO NO NO NO YES NO NOButyric acid Propionic acid YES YES YES NO NO NO NO NO NO NO NO YES NONO YES NO NO Acetoacetic YES YES NO NO NO NO NO NO NO NO NO NO YES NOYES NO NO acid Glucuronamide YES NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO L-Proline NO YES NO NO NO NO NO NO NO YES NO YES NO NO YES NONO D-Xylose YES YES YES YES YES NO NO NO YES NO YES NO NO NO YES NO NOAcetic acid YES YES NO YES NO NO NO YES NO NO YES YES YES NO YES YES NOa-Methyl- YES YES NO NO YES NO NO NO YES NO YES NO NO NO NO NO NOD-Galactoside β-Methyl-D- YES YES NO YES YES NO YES YES YES NO YES NOYES NO YES NO NO glucoside Mucic acid NO YES YES YES YES NO YES YES YESNO NO YES YES NO YES YES NO N-acetyl-β-D- YES YES NO NO YES NO NO NO YESNO YES NO NO NO YES NO YES Mannosamine Pyruvic acid YES YES YES YES YESNO YES NO NO NO YES NO YES NO YES NO NO D-Alanine YES NO NO NO NO NO NONO NO NO NO NO NO NO NO YES NO L-Lactic acid NO YES NO YES YES NO NO NOYES NO NO NO YES NO YES NO NO a-D-Glucose YES YES NO YES YES NO NO NOYES NO YES NO YES NO NO YES YES a-D-Lactose YES YES NO NO NO NO NO YESNO NO YES NO NO NO NO NO NO Adonitol NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO Glycolic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NOYES NO NO Mono Methyl YES YES NO NO NO NO NO NO NO NO YES NO NO NO YESNO NO Succinate L-Galactonic- YES YES YES YES YES NO YES YES YES YES NOYES NO NO YES NO NO acid-?-lactone D-Trehalose YES YES NO YES YES NO NONO YES NO YES NO NO NO NO NO NO Formic acid NO YES NO YES NO NO NO NO NONO YES NO YES NO YES YES NO Maltose YES YES NO YES YES NO YES YES YESYES YES NO YES YES YES NO YES Lactulose YES YES NO NO NO NO NO YES NO NOYES NO NO NO NO NO NO Maltotriose YES YES NO YES YES NO YES YES YES YESYES NO YES YES YES NO YES Glyoxylic acid NO YES YES NO NO NO NO NO NO NONO NO NO NO YES NO NO Methyl YES YES NO NO YES NO YES NO NO YES YES NOYES NO YES NO NO Pyruvate D-Galacturonic NO YES NO YES YES NO NO NO NONO YES YES NO NO YES NO NO acid D-Mannose NO YES NO YES YES NO NO NO YESYES YES NO NO YES NO NO NO D-Mannitol YES YES NO YES YES NO NO YES YESNO YES YES NO NO YES NO NO D-Melibiose YES YES NO YES YES NO NO YES YESNO YES NO NO NO NO NO YES Sucrose YES YES NO YES YES NO NO YES YES NOYES YES NO NO NO NO NO 2-Deoxy NO YES NO YES YES NO YES NO YES NO NO NOYES NO YES NO NO adenosine D-Cellobiose YES YES NO YES YES NO YES YESYES NO YES NO YES YES YES NO YES D-Malic acid NO YES NO NO NO NO NO NONO NO NO NO NO NO YES NO NO Phenylethyl- NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO amine Dulcitol YES NO NO YES NO NO NO NO NO NO NONO NO NO NO NO YES L-Glutamic NO NO NO NO YES NO NO NO NO NO NO NO NO NONO NO NO acid Thymidine YES YES NO YES YES NO YES NO YES NO YES NO YESNO YES NO NO Uridine YES YES YES NO YES NO YES YES YES NO YES YES YES NOYES NO NO Adenosine YES YES YES YES NO NO YES NO NO NO YES NO NO NO YESNO NO Inosine NO YES NO NO NO NO NO NO NO YES YES NO YES NO NO NO NOL-Malic acid NO YES NO NO NO NO NO NO NO NO NO NO YES NO YES NO NO2-Aminoethanol NO NO NO NO NO NO NO NO NO NO YES YES NO NO YES NO NOStrain SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYMSYM Substrate 620 627 628 62C 650 68 70 714 9 905 924 963 978 982 987991 999 D-Serine NO NO YES NO NO NO NO NO NO NO NO NO YES NO NO YES NOD-Glucose- YES YES YES NO NO NO NO YES NO NO NO NO NO NO NO YES NO6-Phosphate L-Asparagine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO L-glutamine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOGlycyl-L- NO NO NO NO YES NO NO NO NO NO NO NO NO NO NO NO NO Asparticacid Glycyl-L- NO NO NO NO NO YES NO NO NO NO NO NO NO NO NO NO NOGlutamic acid Glycyl-L- NO NO NO NO NO YES NO NO NO YES NO NO NO NO NONO NO Proline L-Arabinose NO NO YES NO YES YES YES YES YES NO NO NO NONO YES NO YES D-Sorbitol NO NO NO NO NO NO NO YES NO NO NO NO NO NO NONO NO D-Galactonic NO YES YES NO NO NO NO YES NO NO NO NO NO NO NO NO NOacid-?-lactone D-Aspartic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO m-Tartaric acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO Citric acid NO NO NO NO NO YES NO NO NO NO NO NO NO NO NO NO NOTricarballylic NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO acidp-Hydroxy NO NO NO NO NO NO NO NO NO NO NO NO NO NO YES NO NO Phenylacetic acid N-Acetyl-D- NO YES YES NO NO NO YES YES NO NO NO NO NO NOYES NO NO Glucosamine Glycerol NO NO NO NO NO NO YES YES NO NO NO NO NONO NO NO NO D-L-Malic acid NO NO NO NO NO YES YES YES NO NO NO NO NO YESNO NO NO D-Glucosaminic NO NO NO NO NO NO NO NO YES NO NO NO NO NO NO NONO acid D-Glucose-1- NO NO NO NO NO NO NO YES NO NO NO NO NO NO NO NO NOPhosphate m-Inositol NO NO YES NO NO NO NO YES NO NO NO NO NO NO NO NONO L-Serine NO NO NO NO NO NO NO YES NO NO NO NO NO NO NO NO NOm-Hydroxy YES NO YES NO NO NO NO NO NO NO NO NO NO NO NO NO NO PhenylAcetic acid D-Saccharic NO NO NO NO NO NO YES YES NO NO NO NO NO NO YESNO YES acid L-Fucose NO NO YES NO NO NO YES YES NO NO NO YES YES NO NONO NO D-Ribose NO NO NO NO NO YES YES NO NO NO NO NO NO NO NO NO NO1,2-Propanediol NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOD-Fructose- YES YES YES NO NO YES YES NO NO YES NO NO NO NO NO YES NO6-Phosphate D-Threonine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO L-Threonine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOTyramine NO NO NO NO YES YES NO NO NO NO NO NO NO NO NO NO NO Succinicacid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO D-Glucuronic NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO acid Tween 20 NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO Tween 40 NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO Tween 80 NO NO NO NO NO NO NO YES YES NOYES YES NO NO YES NO NO Fumaric acid NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO L-Alanine YES NO YES NO YES YES YES NO YES NO NO NO NO NONO NO NO D-Psicose NO NO NO NO NO NO YES NO NO NO NO NO NO NO NO NO NOD-Galactose NO NO YES NO NO YES NO NO NO NO NO NO NO NO NO NO NOD-Gluconic YES YES NO NO NO NO YES NO NO NO NO NO NO NO NO NO NO acidL-Rhamnose YES YES YES NO YES YES YES NO NO NO NO NO NO NO NO NO NOa-Keto-Glutaric NO NO NO NO YES YES YES NO NO NO NO NO NO YES NO NO NOacid a-Hydroxy NO NO NO NO YES YES YES NO NO NO NO NO NO NO NO NO NOGlutaric acid- ?-lactone Bromo succinic NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO acid L-Alanyl- NO NO NO NO YES YES NO NO NO NO NO NONO NO NO NO NO Glycine L-Lyxose NO NO NO NO YES YES YES NO NO NO NO NONO NO NO NO NO L-Aspartic NO NO NO NO NO YES YES NO NO YES NO NO NO NONO NO NO acid D-L-a-Glycerol NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO phosphate D-Fructose NO NO NO NO YES YES YES NO NO NO NO NO NONO NO NO NO a-Keto- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOButyric acid a-Hydroxy NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO Butyric acid Propionic acid NO NO NO NO YES NO YES NO NO NO YES YESNO NO NO NO NO Acetoacetic NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO acid Glucuronamide NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO L-Proline NO NO YES NO YES YES NO NO NO NO NO NO NO NO NO NO NOD-Xylose NO YES YES NO YES YES YES YES NO NO NO NO NO NO Acetic acid NONO NO NO NO YES YES NO NO NO NO NO NO YES NO NO NO a-Methyl-D- YES YESYES NO NO NO YES NO NO NO YES YES NO NO NO NO NO Galacto-side β-Methyl-NO YES YES NO NO YES YES NO NO NO NO NO NO NO NO NO NO D-glucoside Mucicacid NO YES YES NO YES YES YES YES NO NO YES YES NO NO YES NO YESN-acetyl-β-D- NO NO YES NO NO NO NO NO NO NO NO NO YES NO NO YES NOMannosamine Pyruvic acid NO YES NO NO YES NO NO NO NO NO NO NO NO NO NONO NO D-Alanine NO NO YES NO NO NO YES NO NO NO NO NO NO NO NO NO NOL-Lactic acid NO NO NO NO NO NO NO YES NO NO NO NO NO NO YES NO NOa-D-Glucose NO NO NO NO YES NO YES YES NO NO NO NO NO NO YES YES NOa-D-Lactose NO NO NO YES NO NO NO YES NO NO NO YES NO NO NO NO NOAdonitol NO NO NO NO NO NO YES YES NO NO NO NO NO NO NO NO NO Glycolicacid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Mono Methyl NONO NO NO YES NO NO NO NO NO YES NO NO NO NO NO YES SuccinateL-Galactonic- YES YES YES NO NO YES YES YES YES NO NO NO NO NO YES NO NOacid-?-lactone D-Trehalose NO NO NO NO YES NO NO YES NO NO NO NO NO NONO NO NO Formic acid NO YES NO NO NO NO YES YES NO NO NO NO NO NO NO NONO Maltose NO YES YES YES YES NO YES YES NO YES NO YES NO NO NO YES NOLactulose NO NO NO YES NO NO NO YES NO NO NO YES NO NO YES NO NOMaltotriose NO YES YES NO NO NO YES YES NO YES NO YES NO NO NO NO NOGlyoxylic acid NO NO NO NO NO NO NO NO NO NO YES NO NO NO NO NO YESMethyl NO NO NO NO NO YES NO NO NO NO NO YES NO NO NO NO YES PyruvateD-Galacturonic YES NO NO NO NO NO NO YES NO NO NO NO NO NO NO NO NO acidD-Mannose NO NO NO NO NO NO NO YES NO YES NO NO NO NO NO NO NOD-Mannitol NO NO NO NO NO NO NO YES NO NO NO NO NO NO NO NO NOD-Melibiose NO YES YES YES NO NO YES YES NO NO NO NO NO NO NO NO NOSucrose NO YES NO NO NO NO NO NO NO NO NO NO NO NO YES YES NO 2-DeoxyYES YES YES NO NO NO YES NO NO NO NO NO NO NO NO NO NO adenosineD-Cellobiose NO YES YES NO NO NO NO YES NO YES NO NO NO NO NO NO NOD-Malic acid NO NO NO NO YES YES NO YES NO NO NO YES NO NO NO NO NOPhenylethyl- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO amineDulcitol NO YES NO YES NO NO NO NO NO NO NO YES NO NO NO NO NOL-Glutamic NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO acidThymidine NO YES YES YES NO NO NO NO NO NO NO YES NO NO NO NO NO UridineNO NO YES NO NO NO YES NO YES NO NO NO NO NO NO NO NO Adenosine YES NOYES NO NO NO NO NO NO NO NO NO NO NO NO NO NO Inosine NO NO NO NO NO NONO YES NO NO NO NO NO NO NO NO NO L-Malic acid NO NO NO NO NO NO NO YESNO NO NO NO NO NO NO NO NO 2-Aminoethanol NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO

TABLE 36D Substrate utilization as determined by BIOLOG PM2A MicroPlatesby culturable bacteria belonging to core OTUs. Strain Substrate SYM103SYM1049 SYM13 SYM17A SYM18 SYM183 SYM184 SYM20 SYM207 SYM212 SYM219SYM234 SYM236 SYM248 SYM249 N-acetyl-D-Galactosamine NO NO NO YES NO YESYES NO NO NO NO NO NO NO NO Gentiobiose NO NO NO YES YES YES YES YES NOYES YES YES NO YES YES D-Raffinose NO NO NO YES NO NO NO NO NO YES YESYES NO YES YES Capric acid NO NO NO NO NO NO NO NO YES NO NO NO NO NO NOD-lactic acid methyl ester NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOAcetamide NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO L-Ornithine YESNO YES YES YES NO YES YES YES YES YES YES YES NO NO Chondrointin sulfateC NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO N-acetyl-neuraminic acidNO NO NO NO NO NO NO NO NO NO NO NO NO NO NO L-glucose NO NO NO NO NO NONO NO NO NO NO NO NO YES NO Salicin NO NO NO YES NO YES YES NO NO YESYES YES NO NO YES Caproic acid NO NO NO NO NO NO NO NO YES NO NO NO NONO NO Malonic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOL-Alaninamide NO NO NO NO NO YES YES NO NO NO NO NO YES NO YESL-Phenylalanine NO NO YES NO NO NO NO NO NO NO NO NO NO NO NOa-Cyclodextrin NO NO NO NO NO NO NO NO NO YES NO YES NO NO NO β-D-alloseNO NO NO YES NO NO NO NO NO NO NO NO NO NO NO Lactitol NO NO NO YES NOYES YES NO NO NO NO YES NO YES NO Sedoheptulosan NO NO NO NO NO NO NO NONO NO NO NO NO NO NO Citraconic acid NO NO YES NO NO NO NO NO NO NO NONO NO NO NO Melibionic acid NO NO NO YES NO NO NO NO NO YES YES NO NOYES NO N-Acetyl-L-Glutamic acid YES NO NO YES NO NO YES NO NO NO NO YESNO NO NO L-Pyroglutamic acid YES NO YES YES YES YES YES YES YES YES YESYES YES YES NO β-Cyclodextrin NO NO NO NO NO NO NO NO NO NO YES YES NONO YES Amygdalin NO NO NO NO NO YES YES NO NO YES NO YES NO YES YESD-Melezitose NO NO NO NO NO NO NO NO NO YES NO YES NO YES YES L-SorboseNO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Citramalic acid NO NO NO NONO NO NO NO NO NO NO NO NO NO NO Oxalic acid NO NO NO NO NO NO NO NO NONO NO NO NO NO NO L-Arginine NO NO NO YES NO NO NO NO NO NO NO NO NO NONO L-Valine YES NO YES YES YES NO YES YES NO YES YES YES NO NO NOγ-Cyclodextrin NO NO NO NO NO NO NO NO NO NO YES YES NO NO NOD-arabinose NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Maltitol NO NONO YES NO YES YES NO NO YES NO YES NO YES YES Stachyose NO NO NO NO NONO NO NO NO YES NO YES NO NO NO D-Glucosamine YES YES YES YES YES YESYES YES NO YES YES YES YES YES YES Oxalomalic acid YES NO YES YES YESYES YES YES NO NO NO YES YES YES YES Glycine NO NO NO NO NO NO NO NO NONO NO NO NO NO YES D,L-Carnitine YES YES YES YES YES NO NO NO NO NO NOYES NO NO YES Dextrin NO NO NO NO NO NO YES NO NO NO YES YES NO NO NOD-arabitol NO NO NO NO NO NO NO NO YES NO NO NO NO NO NOa-Methyl-D-Glucoside NO NO NO NO NO NO NO NO NO NO NO YES YES NO YESD-Tagatose NO NO NO NO NO NO NO NO NO NO NO NO NO NO YES 2-Hydroxybenzoic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Quinic acid NONO NO NO NO NO NO NO NO NO NO NO NO NO NO L-Histidine NO YES NO NO NO NONO NO YES YES NO NO NO NO NO Sec-Butylamine NO NO NO NO NO NO NO NO NONO NO NO NO NO YES Gelatin NO NO NO NO NO YES YES NO YES NO NO YES NO NOYES L-arabitol NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOβ-Methyl-D-Galactoside NO NO NO YES NO NO YES NO NO NO NO YES NO NO NOTuranose NO YES NO YES NO YES YES NO NO YES NO YES NO NO YES 4-Hydroxybenzoic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOD-Ribono-1,4-Lactone NO NO NO YES NO NO NO NO NO NO NO NO NO NO NOL-Homoserine NO NO NO NO NO NO NO NO YES NO NO NO NO NO NOD,L-Octopamine YES NO YES YES YES YES YES YES NO NO YES YES YES NO NOGlycogen NO NO NO NO NO NO NO NO NO NO NO YES NO NO NO Arbutin NO NO NOYES NO YES YES NO NO YES YES YES NO YES YES 3-Methyl Glucose NO NO NO NONO NO NO NO NO NO NO NO NO NO NO Xylitol NO NO NO NO NO NO YES NO NO NONO YES NO NO NO β-Hydroxy butyric acid NO NO NO NO NO NO NO NO NO NO NONO NO NO NO Sebacic acid YES NO NO NO NO NO NO NO NO NO NO NO YES NO NOHydroxy-L-Proline NO NO NO NO NO NO NO NO NO NO NO NO YES NO YESPutrescine YES NO NO YES YES NO NO NO NO YES NO NO NO NO NO Inulin NO NONO YES NO YES YES NO NO NO YES NO NO YES YES 2-Deoxy-D-Ribose NO NO NONO NO NO NO NO NO NO NO NO NO NO NO β-Methyl-D-Glucuronic acid NO NO NONO NO NO NO NO NO NO NO NO NO NO NO N-Acetyl-D-glucosaminitol NO NO NONO NO NO NO NO NO NO NO NO NO NO NO γ-Hydroxy butyric acid NO NO NO NONO NO NO NO NO NO NO NO NO NO NO Sorbic acid NO NO NO NO NO NO NO NO NONO NO NO NO NO NO L-Isoleucine YES NO YES YES NO NO NO NO YES YES NO YESNO NO NO Dihydroxy acetone NO YES NO NO NO NO YES NO NO NO NO NO NO NONO Laminarin NO NO NO NO NO NO NO NO NO NO NO YES NO NO NO i-ErythritolNO NO NO NO NO NO NO NO YES NO NO NO NO NO NO a-Methyl-D-Mannoside NO NONO NO NO NO NO NO NO NO NO NO NO NO NO γ-amino butyric acid YES YES YESYES YES NO NO NO NO NO NO YES NO NO YES a-Keto-valeric acid NO NO NO NONO NO NO NO NO NO NO NO NO NO NO Succinamic acid NO YES NO NO NO NO NONO NO YES NO NO NO NO NO L-Leucine YES NO YES YES NO NO NO NO YES NO NOYES NO NO NO 2,3-Butanediol NO NO YES NO NO NO NO NO NO NO NO YES NO NONO Mannan NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO D-Fucose NO NO NONO NO NO NO NO NO NO NO NO NO NO NO β-Methyl-D-Xyloside NO NO NO YES NONO NO NO NO NO NO YES NO NO NO d-amino valeric acid NO NO NO NO NO NO NONO NO NO NO NO YES NO NO Itaconic acid NO NO NO NO NO NO NO NO YES YESNO NO NO NO NO D-Tartaric acid NO NO NO NO NO NO NO NO NO NO NO NO NO NONO L-Lysine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO 2,3-Butanone NONO NO NO NO NO NO NO NO NO NO NO NO NO NO Pectin NO NO NO NO NO NO NO NONO NO NO YES NO NO NO 3-0-β-D-Galactopyranosyl-D- NO NO NO NO NO NO NONO NO NO NO YES NO NO NO arabinose Palatinose NO NO NO YES NO YES YES NONO YES YES YES NO NO NO Butyric acid NO NO NO NO NO NO NO NO YES NO NONO NO NO NO 5-Keto-D- NO NO NO NO YES NO NO YES NO NO NO NO NO NO NOGluconic acid L-Tartaric acid YES NO YES NO YES NO NO YES NO NO NO NO NONO NO L-Methionine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO3-Hydroxy 2-Butanone NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO StrainSYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM Substrate 260 290 292SYM3 SYM43 SYM50 5066 508 525 SYM53 538A 538B 538i 543 563 574N-acetyl-D-Galactosamine NO NO NO NO NO YES NO NO NO YES NO NO NO NO NONO Gentiobiose YES YES YES NO YES YES NO YES YES YES NO YES YES NO YESNO D-Raffinose YES YES YES NO NO YES NO NO NO YES NO YES NO NO NO NOCapric acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO D-lacticacid methyl ester NO NO NO NO NO YES NO NO NO YES NO NO NO NO NO NOAcetamide NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO YES L-OrnithineYES NO NO YES NO YES NO NO NO NO NO NO NO NO NO NO Chondrointin sulfateC YES NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO N-acetyl-neuraminicacid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO L-glucose NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO Salicin YES YES YES NO NO YES NOYES YES YES NO YES YES NO YES NO Caproicacid YES NO YES NO NO NO NO YESNO NO NO NO NO NO NO NO Malonicacid YES NO NO NO NO NO NO NO YES NO NONO NO NO NO NO L-Alaninamide NO YES NO NO NO NO NO NO YES NO YES NO YESNO NO YES L-Phenylalanine YES NO NO YES NO NO NO NO NO NO NO NO NO NO NOYES a-Cyclodextrin NO YES YES NO NO NO NO NO NO NO YES NO NO YES NO NOβ-D-allose NO NO YES NO NO NO NO NO NO NO NO NO NO NO NO NO Lactitol NOYES YES NO NO NO NO NO YES NO NO YES NO NO NO NO Sedoheptulosan NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO Citraconic acid NO NO NO YES NONO NO NO NO NO NO NO NO NO NO YES Melibionic acid YES NO NO NO NO YES NONO NO YES NO NO NO NO NO NO N-Acetyl-L-Glutamic acid YES NO YES NO NOYES NO NO NO YES NO NO YES NO NO NO L-Pyroglutamic acid YES NO YES YESNO NO NO YES YES NO YES NO YES YES NO YES β-Cyclodextrin NO YES YES NONO NO NO NO NO NO YES YES NO NO NO NO Amygdalin NO YES YES NO NO NO NONO YES NO NO YES YES NO YES NO D-Melezitose NO YES YES NO NO NO NO NOYES NO NO YES NO NO NO NO L-Sorbose NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO Citramalic acid YES NO YES NO NO NO NO NO NO NO NO NO NO NONO NO Oxalic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOL-Arginine YES NO NO NO NO NO NO NO NO NO NO NO NO YES NO YES L-ValineYES NO YES YES NO NO NO NO NO NO NO NO NO NO NO NO γ-Cyclodextrin NO YESYES NO NO NO NO NO NO NO YES YES NO NO NO NO D-arabinose NO YES YES NONO NO NO YES NO NO NO NO NO NO NO YES Maltitol NO YES YES NO NO NO NO NOYES NO NO YES NO NO NO NO Stachyose YES YES YES NO NO NO NO NO YES NO NOYES NO NO NO NO D-Glucosamine YES YES YES YES YES YES NO YES YES YES YESYES YES YES YES YES Oxalomalic acid YES YES YES YES YES NO YES NO YES NOYES YES YES YES YES YES Glycine NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO D,L-Carnitine NO NO NO YES NO NO NO NO NO NO NO NO NO NO NO YESDextrin YES YES YES NO NO NO NO YES YES NO YES YES NO YES NO NOD-arabitol NO NO YES NO NO NO NO YES NO NO NO NO NO NO NO NOa-Methyl-D-Glucoside NO YES YES NO NO NO NO NO NO NO NO YES NO NO NO NOD-Tagatose NO YES NO NO NO NO NO YES NO NO NO NO NO NO NO NO 2-Hydroxybenzoic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Quinic acidNO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO L-Histidine YES YES NONO YES NO NO NO NO NO NO NO NO YES NO YES Sec-Butylamine NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO Gelatin YES YES YES NO NO NO NO NO NONO YES NO YES YES NO NO L-arabitol NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO β-Methyl-D-Galactoside NO YES YES NO NO NO NO YES YES NO NOYES NO NO NO YES Turanose NO YES YES NO NO NO NO NO YES NO NO YES NO NONO NO 4-Hydroxy benzoic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NONO YES D-Ribono-1,4-Lactone NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOYES L-Homoserine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO YESD,L-Octopamine NO NO NO YES NO NO YES NO NO NO NO NO NO NO NO NOGlycogen YES YES YES NO NO NO NO YES NO NO NO YES NO YES NO NO ArbutinNO YES YES NO NO YES NO YES YES YES YES NO YES NO YES NO 3-MethylGlucose NO NO YES NO NO NO NO YES NO NO NO NO NO NO NO NO Xylitol NO NOYES NO NO NO NO NO YES NO NO NO NO NO NO YES β-Hydroxy butyric acid YESNO NO NO NO YES NO YES NO YES NO NO NO NO NO YES Sebacic acid YES NO NONO NO NO NO NO NO NO NO NO NO NO NO YES Hydroxy-L-Proline YES NO YES NONO YES NO NO YES YES NO NO NO YES NO YES Putrescine YES NO NO NO NO YESNO NO NO YES NO NO NO NO NO NO Inulin YES YES YES YES YES NO NO NO NO NOYES YES YES NO NO YES 2-Deoxy-D-Ribose NO NO YES NO NO NO NO YES NO NONO NO NO NO NO NO β-Methyl-D-Glucuronic acid NO NO YES NO NO NO NO NOYES NO NO NO NO NO NO NO N-Acetyl-D-glucosaminitol NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO γ-Hydroxy butyric acid YES NO NO NO NO NO NONO NO NO NO NO NO NO NO NO Sorbic acid NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO L-Isoleucine YES NO YES YES NO NO NO YES NO NO NO NO NONO NO NO Dihydroxy acetone NO NO YES NO NO YES NO YES NO YES NO NO NO NONO YES Laminarin NO YES YES NO NO NO NO NO NO NO NO YES NO NO NO YESi-Erythritol NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOa-Methyl-D-Mannoside NO NO NO NO NO NO NO NO YES NO NO NO NO NO NO NOγ-amino butyric acid YES NO NO YES YES NO NO NO NO NO NO NO NO NO NO YESa-Keto-valeric acid YES NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOSuccinamic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOL-Leucine NO NO YES YES NO NO NO NO NO NO NO NO NO NO NO NO2,3-Butanediol YES NO NO NO NO NO NO NO NO NO NO YES NO NO NO NO MannanNO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO D-Fucose NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO β-Methyl-D-Xyloside NO YES YES NO NO NONO NO NO NO NO YES NO NO NO NO d-amino valeric acid YES NO NO NO NO NONO NO NO NO NO NO NO NO NO NO Itaconic acid YES NO YES NO NO YES NO YESNO YES NO NO NO NO NO NO D-Tartaric acid NO NO NO NO NO NO NO NO NO NONO NO NO NO NO YES L-Lysine YES NO NO NO NO NO NO NO NO NO NO NO NO NONO NO 2,3-Butanone NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOPectin NO YES YES NO NO NO NO YES NO NO NO YES NO NO NO NO3-0-β-D-Galactopyranosyl-D- NO NO YES NO NO NO NO NO NO NO NO YES NO NONO NO arabinose Palatinose NO YES YES NO NO NO NO NO YES NO NO YES NO NONO NO Butyric acid YES NO NO YES NO NO NO NO NO NO NO NO NO NO NO NO5-Keto-D-Gluconic acid NO NO NO NO YES NO NO YES NO NO NO NO NO NO NOYES L-Tartaric acid NO NO NO NO YES NO NO YES NO NO NO NO NO NO NO YESL-Methionine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO 3-Hydroxy2-Butanone NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Strain SYMSYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYMSubstrate 57B 617 620 627 628 62C 650 68 70 714 SYM9 905 924 963 978 982987 991 999 N-acetyl-D-Galactosamine NO NO NO NO YES NO NO NO NO YES NOYES NO NO NO NO NO YES NO Gentiobiose NO YES YES YES YES YES NO NO NOYES NO YES NO NO NO NO NO NO NO D-Raffinose NO NO YES YES YES YES NO NONO YES NO NO NO NO NO NO NO YES NO Capric acid NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO D-lactic acid methyl ester NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Acetamide NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO L-Ornithine NO NO NO NO NO NO NONO YES NO YES NO NO NO NO NO NO NO NO Chondrointin sulfate C NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO N-acetyl-neuraminic acid NONO YES NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO L-glucose NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Salicin NO YES NO YESYES YES NO NO NO YES NO YES NO NO NO NO NO YES NO Caproic acid NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO MaIonic acid NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO L-Alaninamide NO NO NO NONO NO NO NO NO YES NO YES YES NO NO NO NO NO NO L-Phenylalanine NO NO NONO NO NO NO YES NO NO YES NO NO NO NO NO NO NO NO a-Cyclodextrin NO NONO NO NO NO NO NO NO NO NO NO NO NO NO YES NO NO NO β-D-allose NO NO NONO NO NO NO NO NO YES NO NO NO NO NO NO NO NO NO Lactitol NO NO NO NO NOYES NO NO NO YES NO YES NO NO NO NO NO NO NO Sedoheptulosan NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Citraconic acid NO NO NO NONO NO NO YES NO NO YES NO YES NO NO NO NO NO NO Melibionic acid NO NOYES YES YES NO NO YES NO YES NO NO NO NO YES NO NO NO NON-Acetyl-L-Glutamic acid NO NO NO NO NO YES NO NO NO NO NO NO NO NO NONO YES NO NO L-Pyroglutamic acid NO NO NO NO NO YES NO YES YES YES YESNO NO NO NO YES YES NO NO β-Cyclodextrin NO NO NO NO NO NO NO NO NO NONO NO NO NO NO YES NO NO NO Amygdalin NO YES NO NO NO YES NO NO NO NO NONO NO NO NO NO NO YES NO D-Melezitose NO YES NO NO NO YES NO NO NO YESNO NO NO YES NO NO YES NO NO L-Sorbose NO NO NO NO NO NO NO NO NO NO NONO NO YES NO NO YES NO NO Citramalic acid NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO Oxalic acid NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO L-Arginine NO NO NO NO NO NO NO NO NO YES NO NONO NO NO NO NO NO NO L-Valine NO NO NO NO NO NO NO YES YES YES YES NO NONO NO NO NO NO NO γ-Cyclodextrin NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO D-arabinose NO NO NO NO YES NO YES NO NO YES NO NO YESYES NO NO NO NO NO Maltitol NO NO NO YES YES NO NO NO NO YES NO YES NOYES NO NO NO NO NO Stachyose NO NO NO NO NO YES NO NO NO NO NO NO NO YESNO NO NO NO NO D-Glucosamine YES YES NO YES YES YES NO YES YES YES YESYES NO NO YES NO YES YES YES Oxalomalic acid YES YES NO NO NO YES NO YESYES YES YES YES NO NO YES NO YES NO YES Glycine NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO D,L-Carnitine NO NO NO NO NO NO NO NOYES NO YES NO NO NO NO NO NO NO NO Dextrin NO YES YES NO NO YES NO NO NONO NO NO NO NO NO NO NO YES NO D-arabitol NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO a-Methyl-D-Glucoside NO NO NO YES YES NO NONO NO YES NO NO NO YES NO NO NO NO NO D-Tagatose NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO 2-Hydroxy benzoic acid NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO Quinic acid NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO L-Histidine YES NO YES NO NO NOYES NO YES YES NO NO NO NO NO NO NO NO NO Sec-Butylamine NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO Gelatin NO NO NO NO NO NO NONO NO NO NO YES NO NO NO YES NO NO NO L-arabitol NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO β-Methyl-D-Galactoside NO NO YES YESYES YES NO NO NO YES NO NO NO YES YES NO YES NO NO Turanose NO NO NO YESNO YES NO NO NO YES NO NO NO YES NO NO YES NO NO 4-Hydroxy benzoic acidNO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOD-Ribono-1,4-Lactone NO NO NO NO NO NO NO NO NO YES NO NO NO NO NO NO NONO NO L-Homoserine YES NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO D,L-Octopamine NO YES NO NO NO YES NO YES YES NO YES YES NO YES NONO YES YES NO Glycogen NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO Arbutin NO YES NO YES YES YES NO NO YES YES NO YES NO NO YES NONO YES NO 3-Methyl Glucose NO NO NO NO YES NO NO NO NO NO NO NO NO NO NONO NO NO NO Xylitol YES NO NO NO NO NO NO NO YES YES YES NO NO NO NO NONO NO NO β-Hydroxy butyric acid NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO Sebacic acid YES NO NO NO NO NO NO NO NO NO NO NO NONO YES NO NO NO NO Hydroxy-L-Proline NO NO NO NO NO NO YES NO NO NO NONO NO NO NO NO YES YES YES Putrescine NO NO NO NO YES NO NO NO YES NO NONO NO NO NO NO NO NO NO Inulin YES NO NO NO NO YES NO NO NO NO NO NO NONO NO NO YES NO YES 2-Deoxy-D-Ribose NO NO YES NO NO NO YES NO NO NO NONO YES NO NO NO NO NO NO β-Methyl-D-Glucuronic acid NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO N-Acetyl-D-glucosaminitol NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO γ-Hydroxy butyric acidNO NO NO NO NO NO NO NO NO NO NO NO YES NO NO NO NO NO NO Sorbic acid NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO L-Isoleucine NO NONO NO NO YES NO YES YES YES YES NO NO NO NO NO NO NO NO Dihydroxyacetone YES NO NO YES YES NO YES NO NO NO NO NO YES YES NO NO YES NO NOLaminarin NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO YES NO NO NOi-Erythritol NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOa-Methyl-D-Mannoside NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO γ-amino butyric acid YES NO NO NO NO NO YES NO YES YES NO NO NO NONO NO NO NO NO a-Keto-valeric acid NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO Succinamic acid NO NO NO NO NO NO NO NO NO NO NO NOYES NO NO NO NO NO NO L-Leucine YES NO NO NO NO NO NO NO NO YES YES NONO NO NO NO NO NO NO 2,3-Butanediol NO NO NO NO NO YES NO NO NO NO NO NONO NO NO NO NO NO NO Mannan NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO D-Fucose NO NO NO NO NO NO NO NO NO YES NO NO NO NO NO NO NONO NO β-Methyl-D-Xyloside NO NO NO NO NO YES NO NO NO YES NO NO NO NO NONO NO NO NO d-amino valeric acid NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO Itaconic acid NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO YES NO D-Tartaric acid YES NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO L-Lysine NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO 2,3-Butanone NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO Pectin NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOYES NO 3-0-β-D-Galactopyranosyl-D- NO NO NO NO YES NO NO NO NO NO NO NONO NO NO NO YES NO NO arabinose Palatinose NO NO NO YES YES YES NO NO NOYES NO YES NO YES YES NO YES NO NO Butyric acid NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO 5-Keto-D-Gluconic acid NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO L-Tartaric acid NO NO NO NO NONO NO NO YES NO YES NO NO NO NO NO NO NO NO L-Methionine NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO 3-Hydroxy 2-Butanone NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO

TABLE 36E Substrate utilization as determined by BIOLOG PM1 MicroPlatesby culturable fungi belonging to core OTUs. Strain SYM SYM SYM SYM SYMSYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM Substrate 120 122 123124 129 300 310 311 314 315 325 326 327 328 1333 135 136 D-Serine NO NOYES YES NO NO NO YES NO YES NO NO NO NO NO NO NO D-Glucose-6- NO NO NOYES YES NO NO NO YES NO NO NO YES YES NO NO NO Phosphate L-Asparagine NONO NO YES NO YES YES YES YES NO YES YES YES YES YES NO NO L-glutamine NONO NO YES YES YES YES YES YES NO YES YES YES NO YES YES NOGlycyl-L-Aspartic acid NO NO NO NO NO YES NO YES NO YES NO YES n/a NO NONO NO Glycyl-L-Glutamic NO NO NO YES NO YES YES YES YES YES NO NO YESYES NO NO NO acid Glycyl-L-Proline NO NO NO YES NO YES NO YES YES NO NOYES YES YES NO NO NO L-Arabinose YES NO NO NO YES NO YES YES YES YES YESYES YES YES YES YES YES D-Sorbitol NO NO YES YES YES YES YES YES YES YESYES YES YES YES YES YES YES D-Galactonic acid-?- NO NO NO NO NO YES NONO YES NO NO NO YES NO YES NO NO lactone D-Aspartic acid NO NO YES YESNO NO NO YES NO NO NO NO NO NO NO NO NO m-Tartaric acid NO NO NO YES NOYES NO YES YES YES NO NO NO NO NO NO NO Citric acid NO NO NO YES NO YESYES YES YES YES NO YES n/a NO YES NO NO Tricarballylic acid NO NO NO YESNO YES NO YES YES NO YES NO YES YES NO YES NO p-Hydroxy Phenyl NO NO NOYES NO NO NO NO YES NO NO YES YES NO NO NO NO acetic acid N-Acetyl-D- NONO YES YES YES YES YES YES YES YES YES YES YES NO YES YES YESGlucosamine Glycerol YES NO NO YES YES YES YES YES NO YES YES NO YES YESYES NO NO D-L-Malic acid NO NO NO YES NO YES YES YES YES YES NO NO YESYES YES NO NO D-Glucosaminic acid NO NO NO YES NO YES NO NO YES NO NOYES YES NO NO NO NO D-Glucose-1- NO NO YES YES NO YES NO NO YES NO NO NOYES NO NO NO NO Phosphate m-Inositol NO NO NO YES NO YES YES YES YES YESYES YES n/a NO YES NO NO L-Serine NO NO NO YES NO YES YES YES YES NO NOYES YES NO YES NO NO m-Hydroxy Phenyl NO NO NO YES NO YES NO NO NO YESNO NO NO YES NO NO NO Acetic acid D-Saccharic acid NO NO NO YES NO YESYES YES YES YES YES YES YES NO YES NO NO L-Fucose NO NO NO YES NO YES NOYES YES YES NO NO YES NO NO NO NO D-Ribose NO NO YES YES YES YES YES YESNO YES YES YES YES YES NO YES NO 1,2-Propanediol NO NO NO YES NO NO NONO NO NO NO NO NO NO NO NO NO D-Fructose-6- NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO Phosphate D-Threonine NO NO YES YES NO NO NO YESNO NO NO NO n/a YES NO NO NO L-Threonine NO YES NO YES NO YES NO YES NONO NO YES NO YES NO NO NO Tyramine YES NO NO YES NO YES YES YES YES YESNO YES YES NO YES NO NO Succinic acid NO NO NO YES NO YES YES YES NO NONO YES YES NO YES NO NO D-Glucuronic acid NO NO YES YES YES YES YES YESYES YES NO YES YES YES YES YES NO Tween 20 NO NO NO YES YES YES YES YESYES YES NO YES YES NO YES YES YES Tween 40 NO NO YES YES YES YES NO YESYES YES YES YES YES YES YES YES YES Tween 80 NO NO YES NO YES YES YESYES YES YES YES YES YES YES YES YES YES Fumaric acid NO NO YES NO NO YESYES YES YES YES YES YES n/a YES YES NO NO L-Alanine NO NO NO YES NO YESYES YES YES YES YES YES YES YES YES NO NO D-Psicose NO NO NO YES NO NONO NO NO NO NO NO NO YES NO NO NO D-Galactose YES NO YES YES NO YES YESYES YES YES YES YES YES YES YES YES YES D-Gluconic acid NO NO NO YES YESYES YES YES YES NO YES YES YES YES YES YES YES L-Rhamnose NO NO NO YESYES YES NO YES YES YES YES NO YES NO NO NO NO a-Keto-Glutaric acid NO NONO YES NO YES YES YES YES NO NO YES YES YES YES NO NO a-Hydroxy GlutaricNO NO YES YES NO YES YES YES YES NO NO NO YES YES YES NO NOacid-?-lactone Bromo succinic acid NO NO YES YES NO YES YES YES YES NOYES YES n/a NO NO NO NO L-Alanyl-Glycine NO NO YES YES NO YES YES YESYES NO NO YES YES YES YES NO NO L-Lyxose NO NO NO YES NO NO NO NO NO NONO NO NO NO NO NO NO L-Aspartic acid NO NO YES YES NO YES YES YES YESYES NO YES YES YES YES NO NO D-L-a-Glycerol NO NO NO YES NO NO YES NOYES YES NO NO YES NO NO NO NO phosphate D-Fructose NO NO NO YES YES YESYES YES YES YES YES YES YES YES YES YES NO a-Keto-Butyric acid NO NO NOYES NO NO NO YES NO YES NO NO YES NO NO NO NO a-Hydroxy Butyric NO NO NOYES NO NO NO YES NO NO NO NO NO NO NO NO NO acid Propionic acid NO NOYES YES NO YES YES YES YES NO NO NO n/a NO NO NO NO Acetoacetic acid NONO NO YES NO NO NO NO NO YES NO NO NO NO NO NO NO Glucuronamide NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO L-Proline NO NO NO YES NO YESYES YES YES YES YES NO YES YES YES YES NO D-Xylose YES NO NO YES YES YESYES YES YES NO YES NO YES NO YES YES NO Acetic acid NO NO YES YES NO NONO YES NO NO NO NO YES NO NO NO NO a-Methyl-D- NO NO NO YES YES NO YESYES NO NO YES YES YES YES NO YES YES Galactoside β-Methyl-D-glucoside NONO YES YES YES YES YES YES YES NO YES YES NO YES NO YES YES Mucic acidNO NO NO YES YES YES YES YES YES YES NO YES n/a YES YES NO NON-acetyl-β-D- NO NO YES YES NO NO NO NO NO YES NO NO NO YES NO NO NOMannosamine Pyruvic acid NO NO YES YES YES YES YES YES YES NO NO YES YESNO YES NO NO D-Alanine NO NO YES YES NO YES YES YES YES NO NO YES YES NOYES NO NO L-Lactic acid NO NO YES NO NO YES YES YES YES YES NO YES YESNO YES NO NO a-D-Glucose NO NO NO YES YES YES YES YES YES YES YES YESYES YES YES YES YES a-D-Lactose NO NO YES YES YES YES NO YES YES NO NOYES YES YES YES NO NO Adonitol NO NO NO YES NO YES YES YES YES YES YESNO YES NO YES NO NO Glycolic acid NO NO YES YES NO NO NO NO NO NO NO NOn/a NO NO NO NO Mono Methyl NO NO NO YES NO YES NO YES YES NO NO NO YESNO NO NO NO Succinate L-Galactonic-acid-?- NO NO NO NO NO YES YES YESYES NO YES YES YES NO YES YES NO lactone D-Trehalose NO NO NO YES YESYES YES YES YES YES YES YES YES NO YES YES NO Formic acid NO NO NO YESNO NO NO NO NO YES NO NO NO YES NO NO NO Maltose YES NO YES YES YES YESYES YES YES NO YES YES YES YES YES YES YES Lactulose YES NO NO YES NOYES NO YES NO YES NO NO YES YES NO NO NO Maltotriose NO NO YES YES YESYES NO YES YES YES YES YES YES YES NO YES YES Glyoxylic acid NO NO YESYES NO NO NO NO NO NO NO NO n/a NO NO NO NO Methyl Pyruvate NO NO YESYES NO YES YES YES NO NO NO YES YES NO NO NO NO D-Galacturonic acid NONO YES YES YES YES YES YES YES NO NO YES YES NO YES NO NO D-Mannose NONO YES YES YES YES YES YES YES NO YES YES YES YES YES YES YES D-MannitolNO NO YES YES YES YES YES YES YES YES YES YES YES NO YES YES YESD-Melibiose NO NO YES YES YES YES YES YES NO NO YES NO YES YES NO YESYES Sucrose NO NO YES YES YES YES YES YES YES NO YES YES YES YES YES YESYES 2-Deoxy adenosine NO NO NO NO NO NO NO YES NO NO NO NO NO NO NO NONO D-Cellobiose NO NO YES YES NO YES YES YES YES NO YES YES n/a YES NOYES NO D-Malic acid NO NO NO YES NO YES YES YES YES NO NO NO YES NO NONO NO Phenylethyl-amine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO Dulcitol NO NO YES YES YES YES NO YES NO NO NO NO NO YES NO NO NOL-Glutamic acid NO NO YES YES NO YES YES YES YES YES YES YES YES NO YESYES YES Thymidine NO NO NO YES NO NO NO NO NO NO NO NO YES YES NO NO NOUridine NO NO YES YES NO YES YES YES YES NO NO YES YES NO YES NO YESAdenosine NO NO YES YES NO YES NO NO YES YES NO NO YES NO YES NO NOInosine NO NO NO YES YES YES YES YES YES YES NO YES n/a NO YES NO NOL-Malic acid YES NO NO YES NO YES YES YES YES YES YES YES YES NO YES NONO 2-Aminoethanol NO NO YES YES NO YES YES YES YES NO YES NO YES NO YESNO NO Strain SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYMSYM Substrate 151 154 15811 15820 15825 15828 15831 15837 15839 1584715872 15890 15901 15920 15926 15928 D-Serine NO NO NO NO YES NO NO NOYES YES NO NO NO YES NO NO D-Glucose-6- YES YES NO NO NO NO NO NO NO NONO NO NO NO NO NO Phosphate L-Asparagine YES NO YES YES YES YES YES YESNO YES YES NO YES YES YES YES L-glutamine YES NO NO YES YES YES YES YESYES YES YES YES YES YES NO NO Glycyl-L-Aspartic NO YES YES YES YES YESNO NO NO NO NO NO NO YES NO NO acid Glycyl-L-Glutamic NO YES NO YES YESYES NO YES NO YES YES YES YES YES NO NO acid Glycyl-L-Proline YES YES NOYES YES YES YES NO NO YES YES NO YES YES NO YES L-Arabinose YES YES NOYES YES YES YES YES YES YES YES YES YES YES NO YES D-Sorbitol YES YESYES YES YES YES YES YES YES YES YES YES YES YES NO YES D-Galactonicacid- NO YES YES YES NO NO NO YES YES NO NO NO NO YES YES NO ?-lactoneD-Aspartic acid NO YES NO NO NO NO NO YES NO NO NO NO NO YES NO YESm-Tartaric acid YES NO NO NO YES NO YES NO YES NO YES NO YES YES NO NOCitric acid YES YES YES YES YES YES YES YES NO YES NO YES NO NO NO NOTricarballylic acid YES NO NO NO YES NO NO YES YES YES YES YES YES YESNO NO p-Hydroxy Phenyl YES YES YES YES NO YES NO YES YES NO NO NO NO YESNO NO acetic acid N-Acetyl-D- YES YES NO YES YES YES YES NO YES YES YESYES YES YES YES YES Glucosamine Glycerol YES YES YES YES YES YES YES NOYES YES YES YES YES YES YES YES D-L-Malic acid NO YES NO YES YES YES NOYES YES NO YES YES YES YES NO NO D-Glucosaminic NO YES YES NO NO NO NONO NO NO NO YES NO NO NO NO acid D-Glucose-1- NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO Phosphate m-Inositol YES YES NO YES YES YES NO YESYES YES YES YES YES YES NO NO L-Serine NO NO NO YES YES YES NO YES NO NOYES NO YES YES NO NO m-HydroxyPhenyl NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO Acetic acid D-Saccharic acid YES NO NO YES YES YES YES YESYES NO YES YES YES YES NO YES L-Fucose NO YES NO NO NO YES YES NO NO NONO NO NO NO NO NO D-Ribose YES YES NO YES YES YES YES YES YES YES YES NOYES YES NO YES 1,2-Propanediol NO YES NO NO NO YES NO NO NO NO NO NO NONO NO NO D-Fructose-6- NO YES NO NO NO NO NO NO NO NO NO NO NO NO NO NOPhosphate D-Threonine NO NO NO NO NO NO NO YES NO NO NO NO NO YES NO NOL-Threonine NO NO YES YES YES NO NO YES NO NO YES NO YES YES NO NOTyramine YES YES NO NO YES YES NO YES YES YES YES NO YES YES YES NOSuccinic acid YES YES NO YES YES YES NO YES NO NO NO YES YES YES NO NOD-Glucuronic acid NO YES NO YES YES YES NO NO NO YES YES YES YES YES YESYES Tween 20 YES YES YES YES YES YES YES YES YES YES YES NO YES YES YESYES Tween 40 YES YES YES YES YES YES YES YES YES NO YES YES YES YES NOYES Tween 80 YES YES NO YES YES YES YES YES YES YES YES YES YES YES NOYES Fumaric acid NO NO YES YES YES YES YES NO NO NO YES NO YES YES NOYES L-Alanine YES NO YES YES YES YES YES NO NO YES YES YES YES YES YESNO D-Psicose NO YES NO NO NO NO NO NO NO NO NO NO NO NO NO NOD-Galactose YES YES NO YES YES YES YES YES YES YES YES NO YES YES YES NOD-Gluconic acid YES YES YES YES YES YES YES YES YES YES YES YES YES YESNO YES L-Rhamnose YES YES NO NO YES NO NO YES YES YES YES NO YES YES NONO a-Keto-Glutaric NO NO YES YES NO YES NO NO NO NO YES YES NO NO NO NOacid a-Hydroxy Glutaric NO YES NO YES NO YES NO YES NO NO NO NO NO NO NONO acid-?-lactone Bromo succinic acid NO YES NO NO YES YES NO NO YES YESYES NO YES YES NO NO L-Alanyl-Glycine YES YES YES YES YES YES YES YESYES YES YES YES YES YES NO YES L-Lyxose NO NO NO NO NO NO NO NO NO NO NONO NO YES YES NO L-Aspartic acid YES NO NO YES YES YES YES YES NO YESYES YES YES YES NO YES D-L-a-Glycerol NO YES YES YES NO YES NO YES YESNO YES NO NO NO YES NO phosphate D-Fructose YES YES YES YES YES YES YESYES YES YES YES YES YES YES NO NO a-Keto-Butyric acid NO YES NO NO YESNO NO NO NO YES YES NO NO NO NO NO a-Hydroxy Butyric NO YES NO NO YES NONO NO NO NO NO NO NO NO NO NO acid Propionic acid NO YES NO NO YES YESYES YES YES NO NO YES YES YES NO NO Acetoacetic acid NO NO NO NO NO NONO NO NO NO NO NO NO YES NO NO Glucuronamide NO NO YES NO NO NO NO YESNO NO NO NO NO NO NO NO L-Proline YES YES NO YES YES YES YES YES YES YESYES YES YES YES YES YES D-Xylose YES NO NO YES YES YES YES YES YES YESYES NO YES YES NO YES Acetic acid YES YES NO YES YES YES YES YES YES YESYES NO YES YES YES NO a-Methyl-D- YES NO NO NO YES YES YES YES YES YESYES NO YES YES NO NO Galactoside β-Methyl-D- YES YES NO NO YES NO YESYES YES YES YES NO YES YES NO YES glucoside Mucic acid YES YES NO YESYES YES YES YES YES YES YES YES YES YES NO NO N-acetyl-β-D- NO YES NO NONO YES NO YES NO NO NO NO NO NO NO NO Mannosamine Pyruvic acid YES YESNO YES YES YES YES NO YES YES YES YES YES YES NO YES D-Alanine YES NOYES YES NO YES YES YES NO NO NO NO NO YES YES NO L-Lactic acid YES NO NOYES YES YES YES NO YES YES YES NO YES YES NO YES a-D-Glucose YES YES YESYES YES YES YES YES YES YES YES YES YES YES YES YES a-D-Lactose YES NONO NO YES NO NO YES NO YES YES NO YES YES NO NO Adonitol YES YES YES YESYES YES NO YES NO NO YES NO YES YES NO NO Glycolic acid NO YES NO NO YESNO NO NO NO NO NO NO NO NO NO NO Mono Methyl YES NO NO NO YES NO YES NONO NO NO YES NO NO NO YES Succinate L-Galactonic-acid- YES YES NO YESYES YES YES YES YES YES YES YES YES YES YES YES ?-lactone D-TrehaloseYES YES NO YES YES YES YES YES YES YES YES YES YES YES NO YES Formicacid YES NO NO NO YES NO NO NO NO NO YES YES YES NO NO NO Maltose YESYES NO YES YES NO YES YES YES YES YES YES YES YES YES YES Lactulose YESYES NO NO YES NO YES YES NO NO YES NO YES YES NO NO Maltotriose YES NONO YES YES YES YES YES YES YES YES NO YES YES YES YES Glyoxylic acid NOYES NO NO YES NO NO NO NO NO YES NO YES NO NO NO Methyl Pyruvate YES NONO YES YES YES YES YES YES YES YES YES YES YES NO YES D-Galacturonicacid YES YES YES YES YES YES YES YES YES YES YES NO YES YES YES NOD-Mannose YES YES NO YES YES YES YES YES YES YES YES YES YES YES NO YESD-Mannitol YES YES YES YES YES YES YES YES YES YES YES NO YES YES NO YESD-Melibiose YES NO YES YES YES YES YES YES YES YES YES NO YES YES NO YESSucrose YES YES NO YES YES YES YES YES YES YES YES YES YES YES NO YES2-Deoxy adenosine YES NO NO NO YES NO NO NO YES NO YES NO YES NO NO NOD-Cellobiose YES YES NO YES YES NO YES YES YES YES YES YES YES YES NOYES D-Malic acid YES YES YES NO YES NO NO YES YES NO YES NO YES YES NONO Phenylethyl-amine NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NODulcitol YES YES NO NO YES NO YES YES YES YES YES NO YES YES NO YESL-Glutamic acid YES YES YES YES YES YES YES YES YES YES YES YES YES YESNO NO Thymidine NO YES NO NO YES NO YES NO NO NO YES NO YES NO NO NOUridine YES YES YES YES YES NO YES YES NO YES YES YES YES NO NO NOAdenosine NO YES YES YES NO YES YES YES NO YES YES NO YES NO NO NOInosine YES NO YES YES NO YES YES YES NO NO NO YES YES YES YES NOL-Malic acid YES YES NO YES NO YES YES NO NO YES YES NO YES YES NO YES2-Aminoethanol YES YES NO YES YES YES NO YES NO YES YES NO YES YES YESYES Strain SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYM SYMSYM SYM Substrate 15932 160 34 566B 577 590 603 61A 622 629 66 663 696741A 741B 854 880 D-Serine NO NO NO NO NO NO NO YES NO NO NO NO NO NO NONO NO D-Glucose-6- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOPhosphate L-Asparagine NO NO YES NO YES YES YES YES YES YES NO YES YESYES YES YES YES L-glutamine NO YES YES YES YES NO YES YES YES YES NO YESYES YES YES YES NO Glycyl-L-Aspartic acid NO NO YES NO NO NO NO YES NONO NO NO NO NO NO NO NO Glycyl-L-Glutamic NO NO NO NO NO NO YES YES NONO YES NO NO NO YES NO NO acid Glycyl-L-Proline NO YES NO YES YES NO YESYES NO NO NO NO NO NO YES NO NO L-Arabinose YES NO YES NO YES YES YESYES YES NO NO NO YES NO YES YES NO D-Sorbitol NO NO YES NO YES YES YESYES YES YES NO NO NO YES YES NO NO D-Galactonic acid-?- NO NO NO YES NONO YES NO YES NO NO NO NO NO YES NO NO lactone D-Aspartic acid NO NO NONO NO NO NO NO NO NO NO YES NO NO NO YES NO m-Tartaric acid NO NO NO YESYES NO NO YES NO NO NO NO NO NO NO NO NO Citric acid NO NO NO YES YES NOYES YES YES YES NO YES NO YES YES YES NO Tricarballylic acid NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO p-Hydroxy Phenyl NO NO NO YES YESNO YES NO NO NO YES NO NO NO YES NO NO acetic acid N-Acetyl-D- NO NO YESYES YES YES YES YES YES YES NO YES NO NO YES YES NO Glucosamine GlycerolNO NO YES YES NO NO YES YES NO YES YES YES YES YES YES NO NO D-L-Malicacid NO YES NO NO YES NO YES YES YES YES NO YES NO NO YES YES YESD-Glucosaminic acid NO NO YES YES NO NO YES NO NO NO YES NO NO NO YES NONO D-Glucose-1- NO NO NO NO NO NO NO NO NO NO YES NO NO NO NO NO NOPhosphate m-Inositol NO NO NO YES YES NO YES YES YES YES NO NO NO NO YESNO NO L-Serine NO NO NO YES YES NO YES YES YES NO NO YES NO YES YES YESNO m-HydroxyPhenyl NO NO NO NO NO NO NO NO NO NO YES NO NO NO NO NO NOAcetic acid D-Saccharic acid NO NO NO YES YES NO YES YES YES NO NO NOYES NO YES YES NO L-Fucose NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO D-Ribose NO NO YES YES YES YES YES YES YES YES NO NO NO NO YES NONO 1,2-Propanediol NO NO NO NO NO NO NO NO NO NO YES NO NO NO NO NO NOD-Fructose-6- NO NO NO YES NO NO NO NO NO NO NO NO NO NO NO NO NOPhosphate D-Threonine NO NO NO NO NO NO NO YES NO NO NO NO NO NO NO NONO L-Threonine NO NO NO NO YES NO NO YES NO NO NO NO NO NO NO NO NOTyramine YES NO NO NO YES NO YES NO YES YES NO NO NO NO NO NO NOSuccinic acid NO NO YES NO NO NO YES YES YES YES YES NO NO NO YES YES NOD-Glucuronic acid YES NO NO NO YES NO YES YES YES YES NO YES NO YES YESNO NO Tween 20 NO NO YES NO YES YES YES YES NO YES NO NO NO NO YES YESNO Tween 40 NO YES YES YES YES NO YES YES YES YES NO YES NO NO YES YESNO Tween 80 NO NO YES YES YES NO YES YES YES YES YES YES NO NO YES YESNO Fumaric acid NO YES YES YES NO NO YES YES YES YES NO NO NO YES YESYES NO L-Alanine NO NO NO NO YES YES YES YES YES NO YES YES YES YES YESYES NO D-Psicose NO NO NO NO NO NO NO YES NO NO YES NO NO NO NO NO NOD-Galactose NO NO YES NO YES NO YES YES YES NO NO NO YES NO YES YES NOD-Gluconic acid NO NO NO YES YES YES YES YES YES YES NO NO NO YES YESYES NO L-Rhamnose NO NO YES NO YES NO NO YES NO NO NO NO NO NO NO YES NOa-Keto-Glutaric acid NO NO YES NO NO NO YES NO NO YES NO YES NO YES YESYES NO a-Hydroxy Glutaric NO NO NO YES YES NO YES YES YES NO NO NO NO NOYES NO NO acid-?-lactone Bromo succinic acid NO NO YES NO NO NO YES YESNO NO NO NO NO NO YES YES NO L-Alanyl-Glycine NO NO NO NO NO NO YES YESYES YES NO YES NO NO YES YES NO L-Lyxose NO YES NO NO NO NO NO YES NO NONO NO NO NO NO YES NO L-Aspartic acid NO NO YES YES YES NO YES YES YESYES NO YES NO NO YES YES NO D-L-a-Glycerol NO NO NO YES NO NO YES NO YESYES NO YES NO NO YES NO NO phosphate D-Fructose YES YES YES YES YES YESYES YES YES YES NO YES YES NO YES YES NO a-Keto-Butyric acid NO NO NO NONO NO NO YES NO NO NO NO NO NO NO NO NO a-HydroxyButyric NO NO NO NO NONO NO NO NO YES NO NO NO NO NO NO NO acid Propionic acid NO NO NO NO NONO YES NO NO NO NO YES NO NO YES NO NO Acetoacetic acid NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO Glucuronamide NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO L-Proline NO NO YES NO YES NO YES YES YESYES NO YES YES YES YES YES NO D-Xylose NO NO NO YES YES NO YES YES YESYES NO NO YES NO YES YES NO Acetic acid NO NO NO NO NO NO NO NO NO NO NONO NO NO YES NO NO a-Methyl-D- YES NO NO NO YES NO NO YES NO NO NO NO NONO NO NO NO Galactoside β-Methyl-D-glucoside YES YES YES YES YES YES NOYES NO NO YES NO YES NO NO YES YES Mucic acid NO NO NO YES YES NO YESYES YES YES NO YES NO YES YES YES NO N-acetyl-β-D- NO NO NO NO NO NO NOYES NO NO NO NO NO NO NO NO NO Mannosamine Pyruvic acid NO NO NO NO YESNO NO YES NO NO NO YES NO NO YES NO NO D-Alanine NO NO NO YES YES NO YESYES YES YES NO NO NO NO YES YES NO L-Lactic acid NO NO NO NO NO NO YESYES YES NO NO YES YES NO YES NO NO a-D-Glucose YES YES YES YES YES YESYES YES YES YES YES YES YES YES YES YES NO a-D-Lactose NO NO NO NO NO NONO YES NO NO NO YES YES NO NO YES NO Adonitol NO NO NO NO YES NO YES YESYES YES NO YES NO NO YES YES NO Glycolic acid NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO Mono Methyl YES NO NO NO YES NO NO YES NO NO NONO YES NO NO YES NO Succinate L-Galactonic-acid-?- NO NO NO NO YES NOYES YES NO YES NO NO YES NO YES YES NO lactone D-Trehalose YES NO YESYES YES YES YES YES YES YES NO NO YES YES YES YES NO Formic acid NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Maltose YES YES YES NO YESYES NO YES NO NO YES YES YES YES NO YES YES Lactulose NO NO NO NO NO NONO YES NO NO NO YES YES NO NO YES NO Maltotriose YES YES YES NO YES YESNO YES NO NO YES YES YES NO NO YES YES Glyoxylic acid NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO Methyl Pyruvate NO YES NO NO NO NO NOYES NO YES NO YES NO NO YES NO NO D-Galacturonic acid NO NO NO NO YESYES YES YES YES YES YES NO YES NO YES YES NO D-Mannose YES YES YES YESYES NO YES YES YES NO NO YES YES NO YES YES NO D-Mannitol YES NO YES YESYES YES YES YES NO YES YES NO NO NO YES YES NO D-Melibiose NO NO NO NOYES NO NO YES NO NO YES NO YES NO NO YES NO Sucrose YES YES YES YES YESNO YES YES YES YES NO YES YES YES YES YES YES 2-Deoxy adenosine NO NO NONO YES NO NO YES NO NO NO NO NO NO NO NO NO D-Cellobiose YES YES YES NOYES YES NO YES YES NO YES YES YES YES NO YES YES D-Malic acid NO NO NONO YES NO YES YES NO YES NO NO NO NO NO YES NO Phenylethyl-amine NO NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Dulcitol NO NO YES YES YESYES NO YES NO NO YES NO YES NO NO YES NO L-Glutamic acid NO NO YES NO NONO YES YES YES YES NO YES YES NO YES YES NO Thymidine NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO Uridine NO NO NO NO YES NO YES YES YESYES NO NO NO NO YES YES NO Adenosine NO YES NO NO NO NO YES NO YES YESNO NO NO NO YES NO NO Inosine NO NO NO YES YES NO YES YES YES YES NO YESYES NO YES NO NO L-Malic acid NO NO YES NO YES YES YES YES YES NO NO YESYES YES YES YES NO 2-Aminoethanol NO NO NO NO YES NO YES YES YES YES NONO NO NO YES NO NO

TABLE 36F Substrate utilization as determined by BIOLOG PM2A MicroPlatesby culturable fungi belonging to core OTUs. Strain/ SYM- SYM- SYM- SYM-SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- Substrate 120 122123 124 129 1300 1310 1311 1314 1315 1324 1325 1326 1327 1333 N-acetyl-NO NO YES NO NO YES NO NO YES YES NO NO NO YES NO D- Galacto- samineGentio- NO NO YES YES YES NO NO YES NO YES YES YES NO NO biose D- NO NOYES YES YES NO NO YES NO NO NO YES YES NO NO Raffinose Carpice NO NO NONO NO YES NO NO YES NO NO NO YES YES YES acid D-lactic NO YES YES NO NONO NO NO NO NO NO NO NO NO NO acid methyl ester Aceta- NO NO YES NO NONO NO NO NO NO NO NO NO NO NO mide L- NO YES YES YES YES YES YES YES YESYES NO YES YES YES YES Ornithine Chond- NO NO YES NO NO NO NO NO NO YESNO NO NO NO NO rointin sulfate C N-acetyl- NO YES YES NO NO YES NO NOYES NO NO NO NO YES NO neuraminic acid L-glucose NO YES NO NO NO NO NONO NO NO NO NO NO NO NO Salicin YES NO NO YES NO NO NO YES NO NO NO YESYES NO NO Caproic NO NO NO NO NO YES NO NO NO NO NO NO YES NO NO acidMalonic NO NO YES YES NO YES NO NO NO NO NO NO NO NO NO acid L- NO NOYES NO NO YES NO NO YES NO NO YES YES YES YES Alanin- amide L- YES NO NONO NO YES NO YES YES NO NO NO NO YES NO Phenyl- alanine a- YES YES YESYES YES YES YES YES YES YES YES YES YES YES YES Cyclo- dextrinβ-D-allose NO NO YES NO NO NO NO NO NO YES NO NO NO NO NO Lactitol NO NONO NO NO NO NO NO NO YES YES NO NO NO NO Sedohept- YES YES NO YES NO NONO NO NO NO NO NO NO NO NO ulosan Citraconic NO YES YES NO NO NO NO NONO YES NO NO NO NO NO acid Melibionic NO NO NO NO NO NO NO NO YES YES NONO NO NO NO acid N-Acetyl- NO NO NO NO NO YES YES NO YES NO NO NO YESYES YES L-Glutamic acid L- NO NO YES NO NO YES YES YES YES YES NO YESYES YES YES Pyro- glutamic acid β- NO YES YES NO NO NO NO NO NO NO NO NONO NO NO Cyclo- dextrin Amygdalin NO NO NO YES NO NO NO YES NO NO NO YESNO NO NO D- NO YES YES NO YES NO NO YES NO NO YES YES NO NO NOMelezitose L-Sorbose NO NO YES NO YES NO NO NO NO NO NO NO NO NO NOCitramalic YES NO NO NO NO NO NO NO NO NO NO NO NO YES NO acid Oxalicacid NO NO YES YES NO NO NO NO NO NO NO NO NO NO NO L-Arginine NO NO NOYES NO YES YES YES YES YES NO YES YES YES YES L-Valine NO YES YES NO NOYES YES YES YES NO NO YES YES YES YES γ- NO NO YES YES NO NO NO NO NOYES NO YES NO NO YES Cyclo- dextrin D-arabinose NO NO NO YES NO NO NO NONO YES NO NO NO NO NO Maltitol NO NO YES YES YES NO NO YES NO NO YES NONO NO NO Stachyose NO NO NO YES NO NO NO YES NO NO YES YES NO NO NO D-NO NO NO NO NO YES NO NO YES NO NO NO NO YES NO Gluco- samine OxalomalicNO NO YES YES NO NO NO NO NO YES NO NO NO NO NO acid Glycine NO NO NOYES NO NO NO NO NO NO NO NO NO NO YES D,L- NO YES NO NO NO YES NO NO YESNO NO NO YES YES NO Carnitine Dextrin NO NO YES NO YES NO NO YES NO YESNO YES NO NO NO D-arabitol NO YES NO YES NO NO NO YES NO YES YES YES NONO YES a- NO NO NO NO NO NO NO NO NO NO YES NO NO NO NO Methyl-D-Glucoside D-Tagatose NO NO NO YES NO NO NO NO NO NO NO NO NO NO NO2-Hydroxy NO YES NO NO NO NO NO NO YES YES NO NO YES NO NO benzoic acidQuinic acid NO NO NO NO NO YES NO YES YES NO YES YES NO NO NOL-Histidine NO NO NO NO YES YES YES YES YES YES YES NO YES YES YES Sec-NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Butylamine Gelatin YES YESYES YES NO YES YES YES YES YES YES YES YES YES YES L-arabitol NO YES YESNO NO NO NO NO NO YES NO NO NO NO NO β-Methyl- NO NO YES NO NO NO NO YESNO NO NO NO NO NO NO D- Galactoside Turanose NO YES YES YES YES NO YESYES NO NO NO NO NO NO NO 4-Hydroxy NO NO YES NO NO NO NO NO NO YES NO NONO NO NO benzoic acid D-Ribono- NO YES YES NO NO NO NO NO NO YES NO NOYES NO NO 1,4-Lactone L- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOHomoserine D,L- NO NO YES NO NO YES YES YES YES NO NO NO YES YES YESOctopamine Glycogen YES YES YES YES NO YES NO YES NO YES YES YES YES NONO Arbutin NO NO YES YES NO NO NO YES NO YES YES YES NO NO NO 3-MethylYES NO NO NO NO NO NO NO NO NO NO NO NO NO NO Glucose Xylitol NO NO YESNO NO NO NO NO NO YES NO NO NO NO NO β-Hydroxy NO NO YES NO NO YES NOYES NO YES YES YES NO YES YES butyric acid Sebacic acid YES YES YES NONO YES NO NO NO YES NO NO NO NO NO Hydroxy- NO NO NO YES NO YES YES YESYES YES NO YES YES YES YES L-Proline Putrescine NO YES NO YES YES YESYES YES NO YES NO YES NO NO NO Inulin NO YES YES NO NO NO NO NO NO YESNO NO YES NO NO 2-Deoxy- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOD-Ribose β-Methyl-D- YES YES NO NO NO NO NO NO NO YES NO NO NO NO NOGlucuronic acid N-Acetyl-D- NO YES YES NO NO NO NO NO NO NO NO NO NO NONO gluco- saminitol γ-Hydroxy NO YES YES NO NO NO NO NO NO YES NO NO NONO NO butyric acid Sorbic acid NO NO YES NO NO NO NO NO NO NO NO NO NONO NO L-Isoleucine NO NO NO YES YES YES YES YES YES NO NO YES YES YESYES Dihydroxy NO NO YES NO NO NO NO NO NO NO NO NO NO NO NO acetoneLaminarin NO NO NO NO NO NO NO NO NO NO NO YES NO NO NO i-Erythritol NONO YES NO NO NO YES NO NO YES NO NO NO NO YES a-Methyl-D- NO NO YES NONO NO NO NO NO YES NO NO NO NO NO Mannoside γ-amino NO YES NO NO YES YESYES YES YES YES YES YES YES YES YES butyric acid a-Keto- NO NO YES NO NONO NO NO NO YES NO NO NO NO NO valeric acid Succinamic NO NO YES NO NOYES NO NO YES YES NO YES YES YES NO acid L-Leucine NO NO NO YES NO YESYES YES YES YES NO NO YES YES YES 2,3- YES NO YES NO NO NO NO NO NO NONO NO NO NO NO Butanediol Mannan NO NO YES NO NO NO NO NO NO NO NO NO NONO NO D-Fucose NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO β-Methyl- NOYES NO NO NO NO NO NO NO YES NO NO NO NO NO D-Xyloside d-amino NO YESYES NO NO NO NO YES NO YES NO NO NO NO NO valeric acid Itaconic acid NONO YES NO NO NO NO NO NO YES NO NO NO NO NO D-Tartaric NO YES NO NO NONO NO NO NO YES NO NO NO NO NO acid L-Lysine NO NO YES NO NO NO NO YESNO YES NO YES NO NO NO 2,3- NO NO YES NO NO NO NO NO NO YES NO NO NO NONO Butanone Pectin NO YES NO YES NO NO NO YES NO YES NO NO NO NO NO3-0-β-D- NO NO YES NO NO NO YES NO NO YES YES NO NO NO YES Galacto-pyranosyl- D-arabinose Palatinose NO NO YES YES YES NO NO YES NO YES YESYES NO NO NO Butyric acid NO YES NO NO NO NO YES NO NO NO NO NO NO NO NO5-Keto-D- NO NO NO NO NO NO NO YES NO YES NO YES NO NO NO Gluconic acidL-Tartaric NO NO YES NO NO NO NO YES NO NO NO NO NO NO NO acid L- NO YESNO NO NO NO NO NO NO NO NO NO NO NO NO Methionine 3-Hydroxy NO NO NO NONO NO NO NO NO YES NO NO NO NO NO 2-Butanone Strain/ SYM- SYM- SYM- SYM-SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM-Substrate 135 136 151 154 15811 15820 15825 15828 15831 15837 1583915847 15872 15890 15901 15920 15926 N-acetyl-D- NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO Galacto- samine Gentiobiose NO YES YES NO NONO YES NO NO NO YES YES YES NO YES YES NO D-Raffinose YES YES YES YESYES NO YES NO YES YES YES YES YES NO YES YES NO Capric acid NO NO NO NOYES NO NO NO NO NO NO NO NO NO NO NO NO D-lactic acid NO NO NO NO YES NONO NO NO NO NO NO NO NO NO NO NO methyl ester Acetamide NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO L-Ornithine YES NO YES NO NO YES YESYES YES YES YES YES YES YES YES YES YES Chondrointin NO NO NO NO NO NONO NO NO NO NO NO YES NO NO NO NO sulfate C N-acetyl- NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO neuraminic acid L-glucose NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO NO Salicin NO NO YES NO YES NO YESNO YES NO YES YES YES NO YES YES NO Caproic acid NO NO NO NO YES NO NONO NO NO NO NO NO NO NO NO NO Malonic acid NO NO YES NO NO NO YES NO NOYES NO NO YES NO NO NO NO L- NO NO YES NO YES NO YES NO NO NO NO YES YESNO NO NO NO Alanin- amide L- NO NO YES NO NO NO YES NO NO NO YES YES YESNO YES YES NO Phenyl- alanine a- YES YES YES YES YES NO NO YES YES YESYES YES YES YES YES YES YES Cyclo- dextrin β-D-allose NO NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO Lactitol NO NO NO NO YES NO NO NO NO NONO NO NO NO NO NO NO Sedohept- NO NO NO NO NO NO NO NO NO NO NO YES YESNO NO NO NO ulosan Citraconic NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO acid Melibionic NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO acid N-Acetyl- NO NO NO NO NO YES NO YES NO YES NO NO NO NO NO NO YESL-Glutamic acid L- NO YES YES NO YES YES YES YES YES YES YES YES YES YESYES YES NO Pyro- glutamic acid B-Cyclo- NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO dextrin Amygdalin NO NO YES NO NO NO YES NO NO YES YESYES YES NO YES YES NO D- YES YES YES NO YES NO YES NO YES YES YES YESYES NO YES YES NO Melezitose L-Sorbose NO YES YES NO NO NO NO NO NO YESYES NO NO NO YES NO NO Citramalic NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO acid Oxalic acid NO YES NO NO NO NO NO NO NO NO NO NO YES NONO NO YES L-Arginine NO NO YES NO NO YES YES YES YES YES YES YES YES NOYES YES NO L-Valine NO NO NO NO NO YES YES YES NO YES YES NO YES NO YESYES YES γ- NO YES NO NO NO NO YES NO NO NO YES NO YES NO YES YES NOCyclo- dextrin D-arabinose NO NO NO NO NO NO NO NO NO NO NO NO YES NO NONO NO Maltitol NO YES YES NO NO NO YES NO NO NO NO NO YES NO YES YES YESStachyose YES YES YES NO NO NO YES NO YES YES YES YES YES NO YES YES NOD- NO NO NO NO NO NO NO NO YES YES YES NO NO NO NO NO NO GlucosamineOxalomalic NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO acidGlycine NO NO NO NO YES NO YES NO NO NO NO YES YES NO YES NO NO D,L- NONO NO NO NO NO NO NO YES YES NO NO NO NO NO NO NO Carnitine Dextrin YESNO YES NO YES NO YES NO YES YES YES NO YES NO YES YES YES D-arabitol YESNO YES NO NO NO YES NO NO NO NO NO YES NO YES YES NO a-Methyl-D- NO NONO NO NO NO NO NO NO NO YES NO YES NO NO YES NO Glucoside D-Tagatose NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO 2-Hydroxy NO NO NO NO NONO NO NO NO NO NO NO NO NO NO NO NO benzoic acid Quinic acid NO NO YESNO YES NO YES NO NO NO YES NO YES NO NO YES NO L-Histidine NO YES NO NOYES YES YES YES NO NO YES NO YES NO YES YES YES Sec- NO NO NO NO NO NONO NO NO NO NO NO NO NO YES NO NO Butylamine Gelatin NO NO YES NO NO YESYES YES NO YES YES YES YES YES YES YES YES L-arabitol NO NO NO NO NO NONO NO NO NO NO NO YES NO NO NO NO β-Methyl-D- NO YES NO NO NO NO NO NONO YES NO NO YES NO NO NO NO Galactoside Turanose YES YES YES YES NO NOYES NO YES YES YES YES YES NO YES YES NO 4-Hydroxy NO NO NO NO NO NO NONO NO NO NO NO YES NO NO NO NO benzoic acid D-Ribono- NO NO NO NO YES NONO NO NO NO NO NO NO NO NO NO NO 1,4-Lactone L- NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO Homoserine D,L- NO NO NO NO YES YES NO YES NOYES NO NO YES YES NO NO NO Octopamine Glycogen NO YES YES NO NO NO YESNO YES YES YES YES YES NO YES YES NO Arbutin NO YES YES NO NO NO YES NONO YES YES YES YES NO YES YES NO 3-Methyl NO NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO Glucose Xylitol NO NO NO NO YES NO NO NO NO NO NONO YES NO NO NO NO β-Hydroxy NO NO YES NO NO NO NO NO NO NO NO NO NO YESYES YES NO butyric acid Sebacic acid NO NO NO NO NO NO YES NO NO YES NONO YES NO NO NO NO Hydroxy- NO NO YES NO YES YES YES YES YES YES YES YESYES YES YES YES YES L-Proline Putrescine YES YES YES NO NO NO YES YES NONO YES YES YES NO YES YES NO Inulin NO NO NO NO NO NO NO NO NO YES NO NONO YES NO NO NO 2-Deoxy- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO D-Ribose β-Methyl- NO NO NO NO NO NO NO NO NO NO NO NO YES NO NO NOYES D- Glucuronic acid N-Acetyl- NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO D-gluco- saminitol γ-Hydroxy NO NO NO NO YES NO NO NO NO NONO NO NO NO NO NO NO butyric acid Sorbic acid NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO L-Isoleucine NO NO YES NO NO YES YES YES YES YESYES YES YES NO YES YES NO Dihydroxy NO NO NO NO NO NO NO NO NO NO NO NONO NO NO NO NO acetone Laminarin NO NO YES NO NO NO NO NO NO NO YES NONO YES NO YES NO i-Erythritol NO NO NO NO NO NO NO NO NO YES NO NO NO NONO NO NO a-Methyl- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO D-Mannoside γ-amino NO YES YES NO YES NO YES NO YES YES YES YES YES NO YESYES YES butyric acid a-Keto- NO NO NO NO NO NO NO NO NO YES NO NO YESYES NO NO NO valeric acid Succinamic NO NO NO NO NO NO YES NO NO YES NOYES YES NO YES NO NO acid L-Leucine NO NO YES YES NO NO YES NO NO YESYES YES YES NO YES YES NO 2,3- NO NO NO NO NO NO NO NO NO NO NO NO YESNO NO NO NO Butanediol Mannan NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO D-Fucose NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOβ-Methyl- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO D-Xylosided-amino NO NO NO NO NO NO NO NO NO NO NO YES YES NO YES NO YES valericacid Itaconic acid NO NO NO NO YES NO NO NO NO YES NO NO NO NO NO NO YESD-Tartaric NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO acidL-Lysine NO NO YES NO NO NO YES NO YES YES NO YES YES NO YES NO NO 2,3-NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO Butanone Pectin NO NOYES NO NO NO YES YES YES YES YES NO YES NO YES YES YES 3-0-β-D- NO NO NONO NO NO YES NO NO NO NO NO YES NO NO NO NO Galacto- pyranosyl-D-arabinose Palatinose YES YES YES NO YES NO YES NO YES YES YES YES YESYES YES YES NO Butyric acid NO NO NO NO YES NO YES YES NO YES NO NO YESNO YES YES NO 5-Keto-D- NO NO NO NO NO NO NO NO NO NO NO NO NO NO YES NONO Gluconic acid L-Tartaric NO NO YES NO YES NO YES NO NO NO NO NO YESNO YES YES NO acid L- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOMethionine 3-Hydroxy NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO2-Butanone Strain/ SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM-SYM- SYM- SYM- SYM- SYM- SYM- SYM- SYM- Substrate 15928 15932 160 34566B 577 590 603 61A 622 629 66 663 696 741A 741B 854 880 N-acetyl-D- NONO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO YES Galacto- samineGentiobiose YES YES YES YES NO YES YES NO YES NO NO NO NO YES YES NO NONO D-Raffinose YES YES YES NO NO YES NO NO YES NO NO YES NO YES YES NOYES NO Capric acid NO NO NO NO NO NO NO YES NO NO NO NO NO NO NO NO NONO D-lactic acid NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOmethyl ester Acetamide NO NO NO NO NO NO NO NO NO NO NO NO NO YES NO NONO YES L-Ornithine YES YES NO YES YES YES YES YES YES YES NO YES YES YESYES NO NO Chondro- NO YES NO NO NO NO NO NO NO NO NO NO NO YES YES NO NONO intin sulfate C N-acetyl- NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO NO YES neuraminic acid L-glucose NO NO NO NO NO NO NO NO NO NO NONO NO NO YES NO NO NO Salicin YES YES YES YES NO YES YES NO YES NO NO NONO YES NO NO NO NO Caproic acid NO NO NO NO NO NO NO NO NO NO NO NO NOYES NO NO NO NO Malonic acid NO NO NO NO YES NO NO NO YES NO NO NO NO NONO NO NO NO L- NO NO NO YES NO YES NO YES YES NO NO NO NO YES NO YES NOYES Alanin- amide L- NO NO NO NO NO YES NO YES NO NO NO NO NO NO YES NONO NO Phenyl- alanine a- YES YES YES YES YES NO NO YES YES YES NO YES NONO NO YES NO YES Cyclo- dextrin β-D-allose YES NO NO NO NO NO NO NO NONO NO NO NO YES NO NO NO YES Lactitol NO NO NO NO NO NO NO NO YES NO NONO NO YES YES NO NO YES Sedohept- NO NO NO NO NO NO NO NO NO NO NO NO NONO YES NO NO YES ulosan Citraconic NO NO NO NO NO NO NO NO NO NO NO NONO YES NO NO NO YES acid Melibionic NO NO NO NO NO YES NO NO YES NO NONO NO NO YES NO NO NO acid N-Acetyl-L- NO NO NO NO NO NO NO YES NO YESYES NO NO YES NO YES NO YES Glutamic acid L- YES NO NO YES YES YES NOYES YES YES NO NO YES NO YES YES YES YES Pyro- glutamic acid β- NO YESYES NO NO NO NO NO NO NO NO NO NO NO YES NO NO Y Cyclo- dextrinAmygdalin NO YES NO YES NO YES NO NO NO NO NO NO NO NO YES NO YES YES D-YES YES YES YES NO YES NO NO YES NO NO NO NO YES YES NO NO YESMelezitose L-Sorbose NO NO NO NO NO YES NO NO NO NO NO NO NO NO YES NONO NO Citramalic NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO YESacid Oxalic acid NO YES NO NO NO NO NO NO NO NO NO NO NO YES NO NO NOYES L-Arginine YES YES NO YES NO YES NO YES YES YES YES NO YES YES YESYES YES YES L-Valine NO NO NO YES NO YES NO YES NO YES YES NO NO YES YESYES NO YES γ- YES YES YES NO NO YES NO NO YES NO NO NO NO YES NO NO YESNO Cyclo- dextrin D-arabinose NO NO NO NO NO NO NO NO NO NO NO NO NO YESNO NO NO NO Maltitol YES YES YES NO NO YES NO NO YES NO NO NO NO YES YESNO YES YES Stachyose YES YES YES NO NO YES NO NO YES NO NO NO NO YES YESNO YES YES D- NO NO NO YES NO YES NO YES NO YES NO NO NO NO NO NO NO NOGluco- samine Oxalomalic NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO acid Glycine NO NO NO NO NO NO NO NO NO NO NO NO NO YES YES NO NOYES D,L- NO NO NO NO NO NO NO YES NO YES NO NO NO NO NO YES NO NOCarnitine Dextrin YES YES YES YES NO YES NO NO YES NO NO NO NO YES YESNO YES YES D-arabitol YES NO NO YES NO YES NO NO YES NO YES NO NO YESYES NO YES NO a-Methyl- YES NO YES NO NO NO NO NO NO NO NO NO NO NO YESNO NO NO D-Glucoside D-Tagatose NO NO NO NO NO NO NO NO NO NO NO NO YESNO NO NO NO NO 2-Hydroxy NO NO NO NO NO NO NO NO NO NO NO NO NO YES YESNO NO NO benzoic acid Quinic acid NO YES NO YES NO NO NO YES NO NO NO NONO YES NO NO NO YES L-Histidine NO NO NO NO YES YES NO YES YES YES NO NONO YES NO YES NO NO Sec- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NONO NO Butylamine Gelatin YES YES NO YES NO YES YES NO YES NO NO NO NOYES YES YES YES NO L-arabitol NO YES NO NO NO NO NO NO YES NO NO NO NOYES NO NO NO YES β-Methyl-D- NO YES NO YES NO NO NO NO YES NO NO NO NOYES NO NO NO NO Galactoside Turanose YES YES YES YES NO YES NO NO YES NONO NO NO YES NO NO YES YES 4-Hydroxy NO NO NO NO NO NO NO NO YES NO NONO NO YES NO NO NO YES benzoic acid D-Ribono- NO NO NO NO NO NO NO NO NONO NO NO YES YES NO NO YES YES 1,4-Lactone L- NO NO NO NO NO NO NO NOYES NO NO NO NO NO NO NO NO NO Homoserine D,L- NO NO NO NO NO YES NO YESYES YES YES NO NO NO YES YES YES NO Octopamine Glycogen YES YES YES YESNO YES YES NO YES YES NO NO YES YES YES NO YES YES Arbutin YES YES YESYES NO YES YES NO YES NO NO NO NO YES YES YES YES YES 3-Methyl NO NO NONO NO NO NO NO NO NO NO NO NO NO YES NO NO NO Glucose Xylitol NO NO YESNO YES NO NO NO NO NO NO NO NO NO NO NO NO YES β-Hydroxy YES NO NO NOYES YES NO NO NO NO NO NO NO YES YES NO NO NO butyric acid Sebacic acidNO NO NO NO NO NO NO NO YES YES NO NO NO NO NO NO NO NO Hydroxy- NO NONO NO YES YES YES YES YES YES YES NO YES NO YES YES YES YES L-ProlinePutrescine YES NO NO YES NO YES NO YES YES NO NO NO NO YES YES NO NO YESInulin YES NO NO NO NO NO YES NO NO YES YES NO NO YES YES YES NO YES2-Deoxy- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO D-Riboseβ-Methyl- NO YES NO NO NO NO NO NO YES NO NO NO NO YES NO NO NO NO D-Glucuronic acid N-Acetyl- NO NO NO NO NO NO NO NO NO NO NO NO NO YES NONO NO YES D-gluco- saminitol γ-Hydroxy NO NO NO NO NO NO NO NO YES NO NONO NO YES NO NO NO NO butyric acid Sorbic acid NO NO NO NO YES NO NO NOYES NO NO NO NO NO NO NO NO NO L-Isoleucine NO YES NO YES NO YES NO YESNO YES YES NO NO YES YES YES NO YES Dihydroxy NO NO NO NO NO NO NO NO NONO NO NO NO NO NO NO NO NO acetone Laminarin NO NO NO YES NO NO NO NOYES NO NO NO NO NO NO NO NO NO i-Erythritol YES NO NO YES NO NO YES NONO NO YES NO NO YES NO YES YES NO a-Methyl- NO NO NO NO NO NO NO NO NONO NO NO NO YES NO NO NO NO D- Mannoside γ-amino YES NO NO YES NO YESYES YES YES YES YES NO NO YES YES YES YES YES butyric acid a-Keto- NOYES NO NO YES NO NO NO YES NO NO NO NO YES NO NO NO YES valeric acidSuccinamic NO NO NO YES NO NO NO NO YES NO NO NO NO YES YES NO YES YESacid L-Leucine NO NO NO YES NO YES NO YES NO YES YES NO NO NO YES YES NONO 2,3- NO NO NO NO NO NO NO NO YES NO NO NO NO YES NO NO NO YESButanediol Mannan NO NO NO NO NO NO NO NO NO NO NO NO NO YES NO NO NOYES D-Fucose NO YES NO NO NO NO NO NO NO NO NO NO NO YES NO NO NO YESβ-Methyl- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOD-Xyloside d-amino NO NO NO NO NO YES NO NO YES NO NO NO NO NO NO NO NONO valeric acid Itaconic acid NO NO NO NO NO NO NO NO NO NO NO NO NO YESNO NO NO NO D-Tartaric NO NO NO NO NO NO NO NO NO NO NO NO NO YES NO NONO NO acid L-Lysine NO NO NO YES YES NO NO NO YES NO NO NO NO YES YES NONO YES 2,3- NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NOButanone Pectin YES YES YES YES NO YES NO NO YES NO NO NO NO YES YES NOYES YES 3-0-β-D- NO NO NO NO NO YES NO NO NO NO NO NO NO YES YES YES NOYES Galacto- pyranosyl- D-arabinose Palatinose YES YES YES YES NO YESYES NO NO NO NO NO YES YES YES NO YES YES Butyric acid NO NO NO NO NO NONO NO YES NO NO NO NO NO NO NO YES NO 5-Keto-D- NO NO NO NO NO NO NO NONO NO NO NO NO YES NO NO NO NO Gluconic acid L-Tartaric NO YES NO NO NOYES NO NO YES NO NO NO NO NO NO NO NO NO acid L- NO NO YES NO NO NO NONO NO NO NO NO NO NO NO NO NO NO Methionine 3-Hydroxy NO NO NO NO NO NONO NO NO NO NO NO YES NO NO NO NO NO 2-Butanone

Example 9 Testing of Culturable Bacterial and Fungal EndophytesBelonging to Core OTUs on Plants

The results shown above demonstrate that culturable microbes belongingto the same OTUs as microbes core to wheat, soy, cotton, and corn(bacteria) or wheat, cotton, and corn (fungi) possess activities thatcould impart beneficial traits to a plant upon colonization. The aim ofthe experiments in this section addresses the ability of theseculturable bacterial and fungal endophytes to confer beneficial traitson a host plant. Several different methods were used to ascertain this.First, plants inoculated with bacteria or fungi were tested underconditions without any stress to determine whether the microbe confersan increase in vigor. Second, endophyte-inoculated plants were testedunder specific stress conditions (e.g., salt stress or drought stress)to test whether the bacteria confer an increase in tolerance to thesestresses. These growth tests were performed using three different means:using growth assays on water-agar plates; using growth assays on sterilefilter papers; and growth assays in seed germination (rolling) paper.Seeds were treated either with a single bacterial or fungal strain, orwith a combination of two bacterial or two fungal strains.

Seeds and Seed Sterilization

Seeds from soy, corn or wheat were surface-sterilized with chlorine gasand hydrochloric acid as described in Example 5. The seeds were thencoated with bacterial or fungal endophytes as described in Example 5.However, the amount of Sodium Alginate and bacterial suspension orfungal inoculum was adjusted for wheat to 15 ml/kg to account for thelarger surface to volume ratio of these small seeds.

Growth & Scale-Up of Fungi for Inoculation

Fungal isolates were grown from a frozen stock on Petri dishescontaining potato dextrose agar and the plates were incubated at roomtemperature for about a week. After mycelia and spore development, fouragar plugs (1 cm in diameter) were used to inoculate erlenmeyerscontaining 150 ml of potato dextrose broth. Liquid cultures were grownat room temperature and agitation on an orbital shaker at 115 rpm for 4days. Then, the cultures were transferred to 50 ml sterile test tubeswith conical bottoms. Mycelium mats were disrupted by pulse sonicationat 75% setting and 3 pulses of 20 seconds each, using a FisherScientific sonicator (Model FB120) with a manual probe (CL-18). Thesonicated cultures were used in the same manner as the bacterialsuspensions for seed inoculation.

Water Agar Assays

Bacterial or fungal endophytes belonging to OTUs of core microbes weretested for their ability to promote plant growth under normal andsalt-stressed or drought-stressed conditions by inoculating wheat andsoy seeds with those endophytes and germinating them on filter paperand/or water agar

For testing the effect of the endophytes on soy under salt stress,treated soy seeds were placed on Petri dishes (15 cm in diameter) filledwith 50 ml of water agar (1.3% bacto agar) for the normal condition andfilled with 50 ml of water agar with 100 mM NaCl for salt stress. ThreePetri dishes per treatment (each bacterial strain, formulation controlor non treated seeds) and 8 seeds per Petri dish were used. The seedswere placed on the Petri dishes inside a laminar or biosafety hood usingforceps previously flamed. The Petri dishes were sealed with surgicaltape, randomized to avoid position effects and placed in a growthchamber set at 22° C., 60% relative humidity, in the dark for five days.

For testing the effect of salt stress on wheat, a water agar assay wasperformed using square plates 245 mm long on each side (Corning). Eachplate contained 100 ml of water agar (1.3%) for the normal condition andsupplemented with 100 mM NaCl for the salt stress. Two plates were usedper treatment and the seeds were arranged in two rows of 6 seeds eachalong the middle of the plate, with the embryos facing outwardly so thatthe roots would grow from the middle of the plate outwardly and inopposite directions, minimizing roots crossing over among seedlings.

After 5 days of growth, digital images of the seedlings were obtainedand the data scored and analyzed as indicated in Example 5. The effectsof core bacterial and fungal endophytes, alone or in combination, on soyseedlings are shown in Tables 37A and B and 38A and B. The effects ofcore bacterial and fungal endophytes, alone or in combination, on wheatseedlings are shown in Tables 39A and B and 40A and B.

TABLE 37A Assay for soy seedling growth in water agar conditions, wheresoy seeds were treated with core bacterial endophytes. SEQ ID BiologicalBiological Strain NO. Normal Effect? Salt Effect? SYM00009 3589 1 yes 0no SYM00013 3590 0 no — — SYM00018 3592 0 yes — — SYM00020 3593 1 yes —— SYM00053 3601 0 yes 0 yes SYM00062C 3603 0 yes — — SYM00070 3607 0 yes0 yes SYM00103 3609 0 yes 0 yes SYM00184 3621 0 no 1 no SYM00212 3623 2yes 1 yes SYM00234 3625 1 yes 0 yes SYM00249 3628 0 no 0 no SYM00506c3629 1 no 0 yes SYM00507 3630 1 yes 0 no SYM00508 3631 0 yes 0 yesSYM00525 3632 1 no 0 yes SYM00538A 3633 0 no 0 no SYM00538B 3634 0 yes 0no SYM00538i 3635 1 no 0 yes SYM00617 3645 0 yes 0 yes SYM00620 3646 0yes 1 yes SYM00628 3649 0 yes — — SYM00650 3652 0 yes 0 no SYM00714 36560 yes 1 no SYM00905 3663 0 yes 0 yes SYM00924 3664 0 no 0 yes SYM009633665 1 yes 0 no SYM00982 3666 1 yes 0 no SYM00987 3667 0 yes 0 noLegend: 0 indicates <0% effect, 1 indicates <20% effect, 2 indicates<40% effect, 3 indicates >40% effect. For Biological Effect: yesindicates >5% or <−5% effect, no indicates effect between −5% and +5%.

Soy seedlings treated with SYM00009, SYM00020, SYM00212, SYM00234,SYM00506c, SYM00507, SYM00525, SYM00538i, SYM00963, and SYM00982 showedoverall better performance relative to formulation only treated plants.These strains performed up to 40% improvement in normal conditions.Under salt stress conditions, four SYM strains performed up to 20%better relative to formulation only. These data is indicative of thebeneficial effects of the many core bacterial SYM strains on soy underboth normal and biotic stressed conditions.

Based on BIOLOG carbon source assays, SYM00212 that is one of thestrains that conferred beneficial effects on soy are also able to useL-proline as a single source of carbon. It is well established thatendogenous proline level is elevated in plants undergoing drought andsalt stresses (Chen and Murata, 2002). This phenomenon may facilitatemore nutrients to become available for endophytes living symbioticallywith plant hosts exposed to abiotic stresses. Therefore it could bepossible that the ability for endophytes like SYM00212 to scavenge andutilize accumulated amino acids such as proline is associated with itsability to confer beneficial effects on the plant host.

TABLE 37B Assay for soy seedling growth in water agar conditions, wheresoy seeds were treated with combinations of core bacterial endophytes.SEQ ID SEQ ID Biological Salt Biological Strain NO. Strain NO. NormalEffect? stress Effect? SYM00050 3600 SYM00053 3601 2 yes 1 no SYM000503600 SYM00207 3622 1 yes 1 yes SYM00050 3600 SYM00248 3627 1 yes 1 yesSYM00050 3600 SYM00508 3631 1 yes — — SYM00050 3600 SYM00574 3641 1 no 1no SYM00050 3600 SYM00978 3668 0 yes 0 yes SYM00050 3600 SYM00991 3669 1yes 0 no SYM00053 3601 SYM00207 3622 1 yes 1 no SYM00053 3601 SYM002483627 0 no 0 no SYM00053 3601 SYM00508 3631 2 yes 1 yes SYM00053 3601SYM00574 3641 0 yes 0 no SYM00053 3601 SYM00628 3649 1 yes 1 no SYM000533601 SYM00978 3668 — — 0 no SYM00053 3601 SYM00991 3669 2 yes 0 noSYM00053 3601 SYM01049 3671 0 yes 1 no SYM00207 3622 SYM00248 3627 1 yes0 no SYM00207 3622 SYM00508 3631 1 yes 0 no SYM00207 3622 SYM00628 36492 yes 1 no SYM00207 3622 SYM00978 3668 1 no — — SYM00207 3622 SYM009913669 1 yes 1 yes SYM00207 3622 SYM01049 3671 0 yes 1 no SYM00248 3627SYM00574 3641 0 yes — — SYM00248 3627 SYM00628 3649 1 yes 1 no SYM002483627 SYM00991 3669 0 no 1 no SYM00248 3627 SYM01049 3671 0 yes 0 noSYM00508 3631 SYM00574 3641 0 no 0 no SYM00508 3631 SYM00991 3669 1 yes0 yes SYM00508 3631 SYM01049 3671 — — 1 yes SYM00574 3641 SYM00628 36491 yes 1 no SYM00574 3641 SYM00978 3668 1 yes 0 no SYM00574 3641 SYM009913669 1 no 0 no SYM00574 3641 SYM01049 3671 0 yes 0 no SYM00628 3649SYM00978 3668 0 yes 1 no SYM00628 3649 SYM00991 3669 2 yes 0 no SYM009913669 SYM00978 3668 0 yes 0 yes SYM00991 3669 SYM01049 3671 0 yes 0 noSYM01049 3671 SYM00978 3668 0 yes 0 yes Legend: 0 indicates <0% effect,1 indicates <20% effect, 2 indicates <40% effect, 3 indicates <40%effect. For Biological Effect: yes indicates >5% or <−5% effect, noindicates effect between −5% and +5%.

Under normal conditions, 12.8% of core bacteria combinations applied tosoy seeds resulted in >20% improvement in overall seedling phenotypecompared to the formulation-only treatment. In particular, corebacterial strains SYM00053, SYM00207, and SYM00628 each provided twocombinations with other strains that improved the measured phenotypeby >20% as compared to the formulation-only treatment. In combination,these strains seem to synergistically confer benefits towards plantdevelopment. 46.8% of core bacteria combinations resulted in a >5%positive biological effect, and 29.8% of core bacteria combinationsresulted in a >5% negative biological effect as compared toformulation-only treatment, indicating activity of the bacterial strainsin the seed's early developmental stages.

Under salt stress, 14.9% of core bacteria combinations applied to soyseeds resulted in a >5% positive biological effect, and 14.9% of corebacteria combinations resulted in a >5% negative biological effect ascompared to formulation-only treatment, indicating activity of thebacterial strains in the seed's early developmental stages.

TABLE 38A Assay for soy seedling growth in water agar conditions, wheresoy seeds were treated with core fungal endophytes. SEQ ID BiologicalBiological Strain NO. Normal Effect? Salt Effect? SYM00034 3597 0 yes 1no SYM00061A 3602 0 yes 0 yes SYM00066 3605 3 yes 1 yes SYM00120 3610 0yes 0 yes SYM00122 3611 3 yes 0 yes SYM00123 3612 0 yes — — SYM001243613 0 no 1 no SYM00129 3614 0 yes 0 yes SYM00135 3615 3 yes 1 yesSYM00136 3616 1 yes 1 yes SYM00151 3617 1 yes 1 no SYM00154 3618 1 yes 1no SYM00566B 3640 0 yes 2 yes SYM00603 3644 2 yes 1 yes SYM00622 3647 0no 0 yes SYM00629 3650 1 yes 2 yes SYM00663 3654 2 yes 0 yes SYM006963655 0 no 1 yes SYM00741a 3657 0 no 1 yes SYM00741b 3658 2 yes 1 noSYM00793 3659 0 yes 1 no SYM00577 3642 1 yes 1 no SYM00590 3643 2 yes 1yes SYM00854 3661 2 yes 1 yes SYM00880 3662 3 yes 1 no SYM01300 3672 1no 1 no SYM01310 3674 1 yes 1 yes SYM01311 3675 2 yes 1 no SYM01314 36761 no 1 yes SYM01315 3677 3 yes 0 yes SYM01325 3678 0 no 0 no SYM013263679 1 yes 1 yes SYM01327 3680 3 yes 2 yes SYM01328 3681 0 yes 0 yesSYM01333 3682 3 yes 0 yes SYM15811 3683 3 yes 0 yes SYM15820 3684 3 yes1 no SYM15821 3685 3 yes 1 yes SYM15825 3686 2 yes 2 yes SYM15828 3687 2yes 1 yes SYM15831 3688 1 yes — — SYM15837 3689 1 yes 1 yes SYM158393690 2 yes 1 yes SYM15847 3691 — — 1 yes SYM15870 3692 3 yes 1 noSYM15872 3693 3 yes 2 yes SYM15890 3694 3 yes 0 no SYM15901 3695 3 yes 1yes SYM15920 3696 3 yes 1 yes SYM15926 3697 0 yes 1 yes SYM15928 3698 0yes 2 yes SYM15932 3699 3 yes 1 no SYM15939 3700 3 yes 1 yes Legend: 0indicates <0% effect, 1 indicates <20% effect, 2 indicates <40% effect,3 indicates >40% effect. For Biological Effect: yes indicates >5% or<−5% effect, no indicates effect between −5% and +5%.

Of the 53 strains of seed core fungi tested in soybean bioassays, 26(50%) had growth enhancing effects (>20% growth enhancement) onseedlings under normal conditions, while 6 (12%) had growth enhancingeffects under salt conditions.

Fungi SYM00066, SYM00122, SYM00135, SYM00880, SYM01315, SYM01327,SYM01333, SYM15811, SYM15820, SYM15821, SYM15870, SYM15872, SYM15890,SYM15901, SYM15920, SYM15932, and SYM15939 all had strong effects onseedling growth of above 40% under normal conditions. 71% of thesestrains are able to metabolize L-arabinose and Sucrose, while 64% areable to metabolize L-Proline, D-Trehalose and D-Mannose which arecompatible osmolytes produced by plants and microbes under conditions ofwater stress. Under conditions of salt stress, no strain improvedseedling growth more than 40% relative to control, however SYM15872 andSYM01327 which had strong effects under normal conditions had moderateeffects. While not able to dramatically enhance seedling growth undernormal conditions, SYM15932, SYM15825, SYM00629, and SYM00566B were ableto moderately enhance plant growth under conditions of salt stress(20-40%).

TABLE 38B Assay for soy seedling growth in water agar conditions, wheresoy seeds were treated with combinations of core fungal endophytes. SEQID SEQ ID Biological Salt Biological Strain NO. Strain NO. NormalEffect? stress Effect? SYM15901 3695 SYM00124 3613 0 yes 1 yes SYM159013695 SYM15821 3685 0 yes 0 no SYM15901 3695 SYM15870 3692 0 yes 1 noSYM15890 3694 SYM01333 3682 0 yes 0 no SYM15890 3694 SYM00741a 3657 0yes 0 yes SYM15890 3694 SYM00066 3605 0 yes 0 no SYM15890 3694 SYM159013695 2 yes 1 no SYM15890 3694 SYM00124 3613 0 yes 0 yes SYM15890 3694SYM15821 3685 2 yes 0 yes SYM15890 3694 SYM15870 3692 3 yes 1 noSYM15821 3685 SYM15870 3692 3 yes 0 yes SYM15811 3683 SYM00122 3611 0yes 1 no SYM15811 3683 SYM15890 3694 2 yes 0 yes SYM15811 3683 SYM013333682 0 yes 1 no SYM15811 3683 SYM00741a 3657 0 yes 0 no SYM15811 3683SYM00066 3605 0 yes 1 yes SYM15811 3683 SYM15901 3695 0 yes 0 noSYM15811 3683 SYM00124 3613 0 yes 0 yes SYM15811 3683 SYM15821 3685 0yes 1 yes SYM15811 3683 SYM15870 3692 1 yes 1 no SYM01333 3682 SYM00741a3657 0 yes 0 yes SYM01333 3682 SYM00066 3605 0 yes 1 no SYM01333 3682SYM15901 3695 0 no 1 yes SYM01333 3682 SYM00124 3613 0 yes 0 yesSYM01333 3682 SYM15821 3685 0 no 1 yes SYM01333 3682 SYM15870 3692 0 yes1 yes SYM00135 3615 SYM15811 3683 0 yes 1 no SYM00135 3615 SYM00122 36110 yes 1 yes SYM00135 3615 SYM15890 3694 1 yes 1 yes SYM00135 3615SYM01333 3682 0 yes 1 no SYM00135 3615 SYM00741a 3657 0 yes 0 noSYM00135 3615 SYM00066 3605 0 yes 1 no SYM00135 3615 SYM15901 3695 0 yes1 yes SYM00135 3615 SYM00124 3613 0 yes 0 no SYM00135 3615 SYM15821 36850 yes 1 yes Legend: 0 indicates <0% effect, 1 indicates <20% effect, 2indicates <40% effect, 3 indicates >40% effect. For Biological Effect:yes indicates >5% or <−5% effect, no indicates effect between −5% and+5%.

Under normal conditions 5 out of 51 strains (10% of total) conferredbeneficial effect with greater than 20% of growth enhancement includingtwo combinations (SYM15890+SYM15870 and SYM15821+SYM15870) that hadgreater than 40% enhancement in growth. Under salt stress 11 out of 51strains (22% of total) showed significance enhancement (higher than 5%)in growth compared to formulation.

TABLE 39A Assay for wheat seedling growth in water agar conditions,where wheat seeds were treated with core bacterial endophytes. SEQ IDBiological Salt Biological Strain NO. Normal Effect? stress Effect?SYM00003 3588 1 yes 0 yes SYM00009 3589 1 yes 0 yes SYM00013 3590 1 yes0 yes SYM00017A 3591 1 yes 0 yes SYM00018 3592 1 yes 0 yes SYM00020 35932 yes 0 yes SYM00050 3600 2 yes 0 yes SYM00053 3601 1 yes 0 yesSYM00062C 3603 2 yes 1 yes SYM00068 3606 2 yes 0 yes SYM00070 3607 0 no0 yes SYM00103 3609 2 yes 0 yes SYM00183 3620 2 yes 0 yes SYM00184 36212 yes 2 yes SYM00207 3622 1 yes 0 yes SYM00212 3623 2 yes 0 yes SYM002193624 0 no 0 yes SYM00234 3625 1 yes 0 no SYM00236 3626 1 yes 2 yesSYM00248 3627 1 yes 2 yes SYM00249 3628 2 yes 3 yes SYM00506c 3629 1 yes3 yes SYM00507 3630 1 yes 0 yes SYM00508 3631 2 yes 0 yes SYM00525 36322 yes 0 yes SYM00538A 3633 1 yes 0 yes SYM00538B 3634 2 yes 0 yesSYM00538i 3635 1 yes 0 yes SYM00543 3636 1 yes 0 yes SYM00563 3639 2 yes0 yes SYM00574 3641 1 yes 0 yes SYM00617 3645 1 yes 3 yes SYM00620 36462 yes 0 yes SYM00627 3648 2 yes 0 no SYM00628 3649 3 yes 2 yes SYM006503652 1 yes 2 yes SYM00714 3656 1 yes 3 yes SYM00905 3663 1 yes 3 yesSYM00924 3664 1 no 0 yes SYM00963 3665 2 yes 0 yes SYM00978 3668 1 yes 2yes SYM00982 3666 1 yes 0 yes SYM00987 3667 1 yes 3 yes SYM00991 3669 1no 0 yes SYM00999 3670 1 yes 0 yes SYM01049 3671 1 yes 0 yes Legend: 0indicates <0% effect, 1 indicates <20% effect, 2 indicates <40% effect,3 indicates >40% effect. For Biological Effect: yes indicates >5% or<−5% effect, no indicates effect between −5% and +5%.

In general, bacterial endophyte-coated wheat seedlings performed wellcompared to formulation control under normal conditions, i.e., wateragar. 17 out 47 strains tested (36% of total) exhibited move than 20% ofthe enhancement in growth and conferred a significantly noticeablebeneficial effect to wheat seedlings. Under the saline stress condition(water agar supplemented with 100 mM NaCl), 12 out of 44 strains tested(25% of total) exhibited more than 20% of the enhancement in growth,while 6 out of the 47 strains (12% of total) exhibited greater than 40%of the enhancement in growth. Particularly, 3 strains, SYM00184,SYM00249, and SYM00628 conferred a beneficial effect to wheat seedlingsin both normal and saline stress conditions.

TABLE 39B Assay for wheat seedling growth in water agar conditions,where wheat seeds were treated with combinations of core bacterialendophytes. SEQ ID SEQ ID Biological Salt Biological Strain 1 NO. Strain2 NO. Normal Effect? stress Effect? SYM00050 3600 SYM00053 3601 0 yes 0yes SYM00050 3600 SYM00207 3622 2 yes 0 yes SYM00050 3600 SYM00248 36270 no 0 yes SYM00050 3600 SYM00508 3631 3 yes 0 yes SYM00050 3600SYM00574 3641 3 yes 2 yes SYM00050 3600 SYM00978 3668 1 yes 0 yesSYM00050 3600 SYM00991 3669 3 yes 0 yes SYM00053 3601 SYM00207 3622 0 no0 yes SYM00053 3601 SYM00248 3627 2 yes 0 yes SYM00053 3601 SYM005083631 3 yes 0 yes SYM00053 3601 SYM00574 3641 0 yes 0 yes SYM00053 3601SYM00628 3649 2 yes 0 yes SYM00053 3601 SYM00978 3668 1 yes 0 yesSYM00053 3601 SYM00991 3669 1 yes 0 yes SYM00053 3601 SYM01049 3671 1yes 2 yes SYM00207 3622 SYM00248 3627 1 yes 0 yes SYM00207 3622 SYM005083631 0 yes 0 yes SYM00207 3622 SYM00574 3641 2 yes 0 yes SYM00207 3622SYM00628 3649 3 yes 0 yes SYM00207 3622 SYM00978 3668 0 no 0 yesSYM00207 3622 SYM00991 3669 1 yes 0 yes SYM00207 3622 SYM01049 3671 1yes 0 yes SYM00248 3627 SYM00574 3641 2 yes 0 yes SYM00248 3627 SYM006283649 2 yes 0 yes SYM00248 3627 SYM00991 3669 0 no 0 yes SYM00248 3627SYM01049 3671 0 yes 0 yes SYM00508 3631 SYM00574 3641 1 yes 0 yesSYM00508 3631 SYM00628 3649 3 yes 1 yes SYM00508 3631 SYM00991 3669 3yes 0 yes SYM00508 3631 SYM01049 3671 1 no 0 yes SYM00574 3641 SYM006283649 2 yes 0 yes SYM00574 3641 SYM00978 3668 0 no 0 yes SYM00574 3641SYM00991 3669 0 yes 0 yes SYM00574 3641 SYM01049 3671 2 yes 0 yesSYM00628 3649 SYM00978 3668 0 yes 0 yes SYM00628 3649 SYM00991 3669 0yes 0 yes SYM00991 3669 SYM00978 3668 1 yes 0 yes SYM00991 3669 SYM010493671 0 yes 0 yes SYM01049 3671 SYM00978 3668 3 yes 0 yes Legend: 0indicates <0% effect, 1 indicates <20% effect, 2 indicates <40% effect,3 indicates >40% effect. For Biological Effect: yes indicates >5% or<−5% effect, no indicates effect between −5% and +5%.

Under normal condition (water agar), 16 out of 39 (41% of total)combinations of bacterial endophytes conferred a noticeable beneficialeffect with a greater than 20% of growth enhancement. Similarly, undersaline stress condition (water agar supplemented with 100 mM NaCl), 2out of 39 (5% of total) combinations tested conferred a noticeablebeneficial effect with a greater than 20% of growth enhancement.Collectively, there are 3 combinations that conferred an effect in allexperimental conditions. Among the 6 strains in these combinations (3combinations×2), SYM00050, SYM00053, SYM00508, SYM00574, SYM0628 andSYM01049 conferred a beneficial effect to wheat seedlings in both normaland saline stress conditions.

TABLE 40A Assay for wheat seedling growth in water agar conditions,where wheat seeds were treated with core fungal endophytes. SEQ ID SaltStrain NO. Normal stress SYM00034 3597 2 0 SYM00061A 3602 2 0 SYM000663605 2 0 SYM00120 3610 2 0 SYM00122 3611 3 0 SYM00123 3612 1 0 SYM001243613 3 0 SYM00129 3614 3 0 SYM00135 3615 2 0 SYM00136 3616 3 0 SYM001513617 3 0 SYM00154 3618 3 0 SYM00566B 3640 2 0 SYM00577 3642 2 0 SYM005903643 3 0 SYM00603 3644 3 0 SYM00622 3647 3 0 SYM00629 3650 2 0 SYM006633654 3 — SYM00696 3655 2 0 SYM00741a 3657 3 0 SYM00741b 3658 3 0SYM00793 3659 0 0 SYM00854 3661 2 0 SYM00880 3662 2 0 SYM01300 3672 2 0SYM01310 3674 2 0 SYM01311 3675 2 0 SYM01314 3676 3 0 SYM01315 3677 3 0SYM01325 3678 2 0 SYM01326 3679 2 0 SYM01327 3680 3 0 SYM01328 3681 3 0SYM01333 3682 3 0 SYM15811 3683 2 0 SYM15820 3684 2 0 SYM15821 3685 3 0SYM15825 3686 2 0 SYM15828 3687 2 0 SYM15831 3688 2 0 SYM15837 3689 0 0SYM15839 3690 3 0 SYM15847 3691 3 0 SYM15870 3692 3 0 SYM15872 3693 3 0SYM15890 3694 3 0 SYM15901 3695 3 0 SYM15920 3696 3 0 SYM15926 3697 3 0SYM15928 3698 2 0 SYM15932 3699 2 0 SYM15939 3700 1 0 Legend: 0indicates <0% effect, 1 indicates <20% effect, 2 indicates <40% effect,3 indicates >40% effect. For Biological Effect: yes indicates >5% or<−5% effect, no indicates effect between −5% and +5%.

Under the normal condition (water agar), out of the 53 fungal endophytestested, 26 (˜50% of total) conferred a beneficial effect to wheat withgreater than 50% of growth enhancement. These include 7 Fusarium spp., 4Acremonium spp., 4 Alternaria spp., and 4 Cladosporium. Interestingly,these 4 groups of fungi are also top hits in auxin and indolic compoundproduction, acetoin accumulation, and siderophore accumulation (Example5, Table 36B). This suggests a correlation of between the accumulationof auxin, indolic compound, acetoin, and siderophore and the beneficialeffect. Surprisingly, we were unable to identify any fungal strains thatconfer a beneficial effect to wheat in saline stress condition (wateragar supplemented with 100 mM NaCl).

TABLE 40B Assay for wheat seedling growth in water agar conditions,where wheat seeds were treated with combinations of core fungalendophytes. SEQ ID SEQ ID Salt Strain 1 NO. Strain 2 NO. Normal* stressSYM15901 3695 SYM00124 3613 0 − SYM15901 3695 SYM15821 3685 N/A −SYM15890 3694 SYM01333 3682 + N/A SYM15890 3694 SYM00741a 3657 N/A 0SYM15890 3694 SYM01315 3677 0 0 SYM15890 3694 SYM15901 3695 0 − SYM158903694 SYM00124 3613 N/A − SYM15890 3694 SYM15870 3692 0 0 SYM15821 3685SYM15870 3692 N/A − SYM15811 3683 SYM00122 3611 −/−c + SYM15811 3683SYM15890 3694 − + SYM15811 3683 SYM00741a 3657 N/A 0 SYM15811 3683SYM01315 3677 − − SYM15811 3683 SYM00066 3605 0/−c 0 SYM15811 3683SYM15901 3695 +/c − SYM15811 3683 SYM15821 3685 +/c + SYM15811 3683SYM15870 3692 0 + SYM01315 3677 SYM00066 3605 −/−c N/A SYM01315 3677SYM15901 3695 N/A 0 SYM01315 3677 SYM15821 3685 −/−c N/A SYM01315 3677SYM15870 3692 −/−c 0 SYM01333 3682 SYM00741a 3657 −/−d 0 SYM01333 3682SYM01315 3677 −/−c N/A SYM01333 3682 SYM15901 3695 0/−d − SYM01333 3682SYM00124 3613 −/−c, −d N/A SYM01333 3682 SYM15821 3685 −/−d + SYM013333682 SYM15870 3692 N/A 0 SYM00741a 3657 SYM00066 3605 −/−c 0 SYM00741a3657 SYM15901 3695 −/−c, −d − SYM00741a 3657 SYM00124 3613 N/A −SYM00741a 3657 SYM15821 3685 −/−c 0 SYM00741a 3657 SYM15870 3692 −/−c,−d N/A SYM00135 3615 SYM15811 3683 +/d 0 SYM00135 3615 SYM00122 3611−/−e 0 SYM00135 3615 SYM15890 3694 −/d N/A SYM00135 3615 SYM01333 3682−/−c, −d N/A SYM00135 3615 SYM00741a 3657 0/−c N/A SYM00135 3615SYM01315 3677 0 + SYM00135 3615 SYM00124 3613 − − SYM00135 3615 SYM158213685 −/c, d + *Any symbol to the left of the “/” pertains to primaryradicle length with +, 0, − denoting an increase, no change, or decreaserelative to control seedling radicles, respectively. The scale (a-e) tothe right of the “/” pertains to relative increases or decreases insecondary characteristics of the seedlings as follows: a) root hairdevelopment, b) lateral root number, c) lateral root size, d) shootlength, and e) root thickness.

Under normal condition (water agar), four combinations of core fungiendophytes conferred a noticeable beneficial effect with longer rootgrowth relative to formulation only treated seeds. Under saline stresscondition (water agar supplemented with 100 mM NaCl), seven combinationsthat were tested conferred noticeable beneficial effect with a greaterroot length in comparison with formulation only treated wheat seeds.

Filter Paper Growth Assay

Wheat seeds were sterilized and coated with the appropriate endophyte asdescribed in Example 7. They were then placed in filter paper growthassays as described in Example 5. After 5-8 days of growth, a picture ofeach plate was taken and analyzed as described in Example 5.

The effects of bacterial and fungal endophytes belonging to core OTUs onthe growth of wheat seeds in a filter paper assay is shown in Tables 41Aand B and 42A and B.

TABLE 41A Growth of wheat seeds treated with bacterial endophytesbelonging to OTUs present in corn, wheat, cotton and soy seeds. SEQ IDBiological Water Biological Strain NO. Normal Effect? stress Effect?SYM00003 3588 1 yes — — SYM00009 3589 1 no 1 yes SYM00013 3590 1 yes 1yes SYM00017A 3591 0 no 1 yes SYM00018 3592 0 yes 1 no SYM00020 3593 1yes 1 no SYM00050 3600 0 no 0 no SYM00053 3601 1 yes 1 yes SYM00062C3603 0 yes 2 yes SYM00068 3606 2 yes 0 no SYM00070 3607 1 yes 1 yesSYM00103 3609 0 no 1 yes SYM00183 3620 1 no 1 yes SYM00184 3621 1 yes 0no SYM00207 3622 0 no 1 no SYM00212 3623 0 yes 2 yes SYM00219 3624 0 no0 yes SYM00234 3625 1 yes 1 yes SYM00236 3626 1 yes 1 yes SYM00248 36272 yes 1 yes SYM00249 3628 2 yes 0 no SYM00506c 3629 2 yes 1 yes SYM005073630 1 yes 0 yes SYM00508 3631 2 yes 1 yes SYM00525 3632 1 yes 1 yesSYM00538A 3633 2 yes 0 no SYM00538B 3634 0 no 2 yes SYM00538i 3635 0 yes2 yes SYM00543 3636 1 yes 0 yes SYM00563 3639 1 yes 1 no SYM00574 3641 0yes 0 no SYM00617 3645 2 yes 0 yes SYM00620 3646 2 yes 1 yes SYM006273648 2 yes 0 yes SYM00628 3649 1 yes 2 yes SYM00650 3652 1 yes 0 yesSYM00714 3656 0 yes 1 yes SYM00905 3663 2 yes 3 yes SYM00924 3664 2 yes0 yes SYM00963 3665 1 yes 0 no SYM00978 3668 3 yes 0 no SYM00982 3666 0yes 0 yes SYM00987 3667 1 yes 0 no SYM00991 3669 1 no 1 yes SYM009993670 1 yes 1 yes SYM01049 3671 0 no 1 yes Legend: 0 indicates <0%effect, 1 indicates <20% effect, 2 indicates <40% effect, 3indicates >40% effect. For Biological Effect: yes indicates >5% or <−5%effect, no indicates effect between −5% and +5%.

Under the normal condition (filter paper soaked with sterile water), 12out of 32 tested strains (38% of total) conferred beneficial effect towheat with greater than 20% of growth enhancement, whereas 6 out of 43strains (14% of total) tested showed beneficial effect under waterstress condition (filter paper soaked with 8% PEG 6000). The bacterialendophytes treated on wheat seeds and tested in filter paper assays thatproduced beneficial effects belong to a great diversity of genera:Pseudomonas, Curtobacterium, Paenibacillus, Bacillus, Enterobacter,Agrobacterium, Chrysobacterium, Escherichia and Methylobacterium. Wehave not observed over-representation of certain taxonomic groups ofbacteria. All of these bacteria produced intermediate to high levels ofauxin, acetoin and siderophore production (Example 5, Table 34A) and areable to metabolize intermediate to large numbers of substrates (Table31B). The exceptions to this were SYM00982 that had low levels ofsiderophore production and only metabolized 3 substrates and SYM00999that had low levels of both siderophore and auxin production andmetabolized 6 substrates.

TABLE 41B Growth of wheat seeds treated with combinations of bacterialendophytes, belonging to OTUs present in corn, wheat, cotton and soyseeds. SEQ SEQ ID ID Biological Water Biological Strain 1 NO. Strain 2NO. Normal effect? stress effect? SYM00053 3601 SYM01049 3671 1 No 0 YesSYM00207 3622 SYM00248 3627 2 Yes 0 Yes SYM00207 3622 SYM00508 3631 2Yes 0 Yes SYM00207 3622 SYM00574 3641 1 Yes 0 Yes SYM00207 3622 SYM006283649 1 Yes 1 Yes SYM00207 3622 SYM00978 3668 1 yes 0 Yes SYM00207 3622SYM00991 3669 1 Yes 0 Yes SYM00207 3622 SYM01049 3671 2 Yes 1 NoSYM00248 3627 SYM00574 3641 0 Yes 0 Yes SYM00248 3627 SYM00628 3649 2Yes 0 Yes SYM00248 3627 SYM00991 3669 2 Yes 1 No SYM00248 3627 SYM010493671 2 Yes 0 Yes SYM00508 3631 SYM00574 3641 2 Yes 0 Yes SYM00508 3631SYM00628 3649 0 Yes 0 Yes SYM00508 3631 SYM00991 3669 0 Yes 0 YesSYM00508 3631 SYM01049 3671 1 No 0 Yes SYM00574 3641 SYM00628 3649 1 Yes0 Yes SYM00574 3641 SYM00978 3668 1 Yes 0 Yes SYM00574 3641 SYM009913669 0 No 0 Yes SYM00574 3641 SYM01049 3671 1 yes 0 Yes SYM00628 3649SYM00978 3668 1 Yes 0 Yes SYM00628 3649 SYM00991 3669 3 Yes 0 YesSYM00991 3669 SYM00978 3668 1 Yes 0 Yes SYM00991 3669 SYM01049 3671 1Yes 0 Yes SYM01049 3671 SYM00978 3668 1 No 0 Yes 0 indicates <0% effect,1 indicates <20% effect, 2 indicates <40% effect, 3 indicates >40%effect. For Biological Effect: yes indicates >5% or <−5% effect, noindicates effect between −5% and +5%.

A variety of binary combinations of bacterial endophytes conferred abenefit under non-stress and/or water stress conditions. SYM00207, forinstance, is present in several combinations that provide a benefitunder normal conditions, including in tandem with SYM00574 or SYM01049.None of these strains alone confer a benefit to normal condition plants.SYM00628 provides an observable benefit under both normal andwater-limited conditions. The benefit under normal conditions isincreased when SYM00628 is combined with SYM00991, which itself alsoprovides a relatively lower benefit under the normal condition. SYM00628belongs to the genus Enterobacter, while SYM00991 belongs to the genusAcidovorax.

TABLE 42A Growth of wheat seeds treated with fungal endophytes belongingto OTUs present in corn, wheat, and cotton seeds SEQ ID Water Strain NO.Normal stress SYM00034 3597 1 0 SYM00061A 3602 1 0 SYM00066 3605 2 0SYM00120 3610 1 1 SYM00122 3611 0 0 SYM00123 3612 0 0 SYM00124 3613 2 0SYM00129 3614 0 0 SYM00135 3615 2 1 SYM00136 3616 2 0 SYM00151 3617 2 2SYM00154 3618 2 2 SYM00566B 3640 1 0 SYM00603 3644 1 0 SYM00622 3647 2 3SYM00629 3650 0 0 SYM00663 3654 0 0 SYM00696 3655 0 0 SYM00741a 3657 1 0SYM00741b 3658 0 0 SYM00793 3659 1 0 SYM00577 3642 1 0 SYM00590 3643 1 0SYM00854 3661 2 0 SYM00880 3662 2 0 SYM01300 3672 0 0 SYM01310 3674 1 0SYM01311 3675 0 0 SYM01314 3676 2 0 SYM01315 3677 1 0 SYM01325 3678 2 0SYM01326 3679 2 1 SYM01327 3680 2 2 SYM01328 3681 2 0 SYM01333 3682 1 0SYM15811 3683 0 2 SYM15820 3684 2 2 SYM15821 3685 2 0 SYM15825 3686 2 1SYM15828 3687 2 0 SYM15831 3688 2 0 SYM15837 3689 0 0 SYM15839 3690 2 0SYM15847 3691 2 0 SYM15870 3692 2 0 SYM15872 3693 2 0 SYM15890 3694 3 0SYM15901 3695 2 2 SYM15920 3696 2 0 SYM15926 3697 2 2 SYM15928 3698 0 0SYM15932 3699 2 0 SYM15939 3700 1 0

Under the normal condition (filter paper with sterile water), out of the53 fungal endophytes tested, 34 (˜61% of total) conferred a beneficialeffect to wheat with greater than 20% of growth enhancement. Severaltaxonomic groups of fungi, including Fusarium, Acremonium, Alternaria,and Cladosporium, are over-represented. Interestingly, these 4 groups offungi are also top hits in auxin and indolic compound production,acetoin accumulation, and siderophore accumulation (Example 5, Table36B). This suggests a correlation of between the accumulation of auxin,indolic compound, acetoin, and siderophore and the beneficial effect.Under the water stress condition (filter paper with 8% PEG 6000), 8 outof 53 strains tested (15% of total) conferred a beneficial effect towheat with greater than 20% of growth enhancement. Similarly, the tophits belong to the genera of Fusarium, Alternaria, Acremonium, andCladosporium. However, not over-representation of these taxonomic groupshas been observed. Of all strains tested, 9 strains, including 3Alternaria spp. and 2 Fusarium spp., conferred a beneficial effect towheat in both normal and water stress conditions.

TABLE 42B Growth of wheat seeds treated with combinations of fungalendophytes belonging to OTUs present in corn, wheat, and cotton seeds.SEQ ID SEQ ID Biological Water Biological Strain 1 NO. Strain 2 NO.Normal effect? stress effect? SYM15901 3695 SYM00124 3613 1 No 0 yesSYM15901 3695 SYM15821 3685 0 Yes 0 No SYM15901 3695 SYM15870 3692 2 Yes1 Yes SYM15890 3694 SYM01333 3682 1 Yes 1 Yes SYM15890 3694 SYM00741a3657 1 Yes 0 No SYM15890 3694 SYM01315 3677 1 Yes 1 no SYM15890 3694SYM00066 3605 1 Yes 0 Yes SYM15890 3694 SYM15901 3695 1 Yes 2 YesSYM15890 3694 SYM00124 3613 0 No 2 Yes SYM15890 3694 SYM15821 3685 1 Yes0 Yes SYM15890 3694 SYM15870 3692 0 Yes 0 Yes SYM15821 3685 SYM158703692 2 yes 0 Yes SYM15811 3683 SYM00122 3611 1 Yes 0 Yes SYM15811 3683SYM15890 3694 1 Yes 0 No SYM15811 3683 SYM01333 3682 2 Yes 1 No SYM158113683 SYM00741a 3657 1 Yes 1 No SYM15811 3683 SYM01315 3677 1 Yes 0 yesSYM15811 3683 SYM00066 3605 1 Yes 0 No SYM15811 3683 SYM15901 3695 1 No0 No SYM15811 3683 SYM00124 3613 0 No 1 No SYM15811 3683 SYM15821 3685 1Yes 1 Yes SYM15811 3683 SYM15870 3692 1 No 1 Yes SYM01315 3677 SYM000663605 2 Yes 1 Yes SYM01315 3677 SYM15901 3695 2 Yes 2 Yes SYM01315 3677SYM00124 3613 1 Yes 0 yes SYM01315 3677 SYM15821 3685 2 Yes 2 yesSYM01315 3677 SYM15870 3692 0 No 1 No SYM01333 3682 SYM00741a 3657 1 No0 Yes SYM01333 3682 SYM01315 3677 1 Yes 0 yes SYM01333 3682 SYM000663605 0 Yes 1 Yes SYM01333 3682 SYM15901 3695 0 Yes 1 Yes SYM01333 3682SYM00124 3613 1 Yes 0 Yes SYM01333 3682 SYM15821 3685 0 Yes 0 YesSYM01333 3682 SYM15870 3692 1 No 0 Yes SYM00741a 3657 SYM01315 3677 0 No1 no SYM00741a 3657 SYM00066 3605 1 Yes 0 No SYM00741a 3657 SYM159013695 1 Yes 2 Yes SYM00741a 3657 SYM00124 3613 0 No 0 Yes SYM00741a 3657SYM15821 3685 1 No 2 Yes SYM00741a 3657 SYM15870 3692 2 Yes 1 YesSYM00135 3615 SYM15811 3683 1 Yes 0 No SYM00135 3615 SYM00122 3611 1 Yes0 Yes SYM00135 3615 SYM15890 3694 1 Yes 2 Yes SYM00135 3615 SYM013333682 1 Yes 0 Yes SYM00135 3615 SYM00741a 3657 2 Yes 0 Yes SYM00135 3615SYM01315 3677 1 Yes 0 yes SYM00135 3615 SYM00066 3605 2 Yes 1 NoSYM00135 3615 SYM15901 3695 1 Yes 1 Yes SYM00135 3615 SYM00124 3613 2Yes 0 No SYM00135 3615 SYM15821 3685 0 No 1 Yes Legend: 0 indicates<0%effect, 1 indicates <20% effect, 2 indicates <40% effect, 3indicates >40% effect. For Biological Effect: yes indicates >5% or <−5%effect, no indicates effect between −5% and +5%.

Several core combinations of fungal SYM strains showed beneficialeffects on wheat both under normal and water stress conditions. Thecombinatory effect of these beneficial strains was indicative of overallsynergistic underlying mechanisms that drive beneficial effect on theseedlings treated with those strains. In particular, the combination ofSYM01315+SYM15901 showed up to 40% beneficial effect on wheat plants inboth normal and water stressed conditions relative to formulation onlytreated wheat seedlings. SYM01315 is able to utilize L-proline as a solecarbon substrate based on BIOLOG assays. Similarly, proline was also asole carbon source for SYM15901. Taken together, it is highly suggestivethat the SYM strains' ability to efficiently utilize proline that getselevated under biotic stresses as water and salt is a potential keycomponent in conferring water stress plant protection.

Rolling Paper Assay for Evaluating Seed Germination and Seedling DroughtTolerance

Soy seeds were sterilized and coated with the appropriate endophyte asdescribed in Example 5. Regular weight seed germination paper (AnchorPaper Co.) was used for testing the effect of the endophytes on soyunder water limiting stress. Briefly, the paper was custom cut to 60cm×15 cm and soaked in sterile water. Sixteen seeds were placed alongthe middle (7.5 cm from the edge) of longest axis of a piece of paper,equidistant from one another. A second pre-soaked piece of paper waslayered on top of the seeds and the “sandwich” created in this way wasrolled from one end, being careful to maintain the seeds in position, toform a tube 15 cm tall and approximately 5 cm in diameter. Each paperroll was placed vertically inside a sterile glass jar with a lid to holdwater absorbed in rolling paper, and was then incubated in a growthchamber set at 22° C., 60% relative humidity, in the dark for two days.Then, the jars were opened and incubated for two more days with theconditions changed to 12 hours daylight (300-350 micro Einstein) 12hours dark and 70% relative humidity.

Data scoring and analyzes were performed as described in Example 5,except for experiment shown in Table 44A, where measurements were takenby hand, and the data are showed in a binning of % increase in rootlength vs fungal formulation control: >0%=0, 0-5%=1, 5-10%=2, 10-15%=3.

The effects of bacterial and fungal endophytes belonging to core OTUs,and combinations of bacterial and fungal endophytes, on the growth ofsoy seeds in a rolling paper assay is shown in Tables 43 and 44A and B.

TABLE 43A Assay for soy seedling growth in rolling paper assay, wheresoy seeds were treated with core bacterial endophytes. Measurements formanual scoring of rolling paper soy water stress assay for core fungalstrains were done according to the scale established previously. Brieflythe score that was used was: <0% = 0; >0% = 1; >5% = 2, and >10% = 3.The percentage indicates percent change of treatments relative toformulation. The mean root lengths of the SYM treated biologicalreplicates of soy seedlings were divided relative the mean root lengthsof the fungal formulation. SEQ ID Water Strain NO. stress SYM00003 35880 SYM00013 3590 3 SYM00017A 3591 3 SYM00018 3592 2 SYM00020 3593 2SYM00050 3600 1 SYM00070 3607 3 SYM00219 3624 2 SYM00525 3632 3SYM00538A 3633 3 SYM00538i 3635 3 SYM00627 3648 3 SYM00987 3667 3SYM00991 3669 1 SYM00628 3649 1

Under water stress, 73.33% of bacterial strains showed beneficial effectcompared to control higher than 20% including SYM00018, SYM00020 andSYM00219. Strains SYM00013, SYM00017A, SYM00070, SYM00525, SYM00538A,SYM00538i, SYM00627, SYM00987 (53.3% of total) showed beneficial effecthigher than 40%.

TABLE 44A Assay for soy seedling growth in rolling paper assay, wheresoy seeds were treated with core fungal endophytes. Measurements formanual scoring of rolling paper soy water stress assay for core fungalstrains were done according to the scale established previously. Brieflythe score that was used was: <0% = 0; >0% = 1; >5% = 2, and >10% = 3.The percentage indicates percent change of treatments relative toformulation. The mean root lengths of the SYM treated biologicalreplicates of soy seedlings were divided relative the mean root lengthsof the fungal formulation. SEQ ID Water Strain NO. stress SYM00066 36050 SYM00122 3611 3 SYM00123 3612 2 SYM00124 3613 3 SYM00135 3615 3SYM00741a 3657 0 SYM00741b 3658 3 SYM00795 3660 — SYM00854 3661 0SYM00880 3662 1 SYM01303 3673 0 SYM01315 3677 3 SYM01327 3680 2 SYM013333682 3 SYM15811 3683 3 SYM15820 3684 1 SYM15821 3685 2 SYM15831 3688 3SYM15870 3692 1 SYM15872 3693 0 SYM15890 3694 3 SYM15901 3695 3 SYM159203696 0 SYM15932 3699 3 SYM15939 3700 0

Water stress experiments examining the effect of core fungal SYM strainson soy plants revealed several very promising strains that thatexhibited over 10% improved seedling root length relative to formulationonly treated seedlings. These were SYM00122, SYM00124, SYM00135,SYM00741b, SYM01315, SYM01333, SYM15811, SYM15831, SYM15890, SYM15901,and SYM15932. Three strains had >5% improved root length, while threeshowed >0% increased root length compared to formulation only treatedplants.

Eight of these eleven had the ability to utilize L-proline as a solecarbon nutrient in BIOLOG substrate tests. It is well established thatproline level is elevated in plants exposed to salt and water stresses.This data may indicate that although the SYM strains originated fromdifferent genera, they may share similar underlying mechanisms when itcomes to affecting the plants phenotype in mitigating plant droughtstress.

TABLE 44B Assay for soy seedling growth in rolling paper assay, wheresoy seeds were treated with combinations of core fungal endophytes.Measurements for manual scoring of rolling paper soy water stress assayfor core fungal strains were done according to the scale establishedpreviously. Briefly the score that was used was: <0% = 0; >0% = 1; >5% =2, and >10% = 3. The percentage indicates percent change of treatmentsrelative to formulation. The mean root lengths of the SYM treatedbiological replicates of soy seedlings were divided relative the meanroot lengths of the fungal formulation. SEQ ID SEQ ID Water Strain 1 NO.Strain 2 NO. stress SYM15901 3695 SYM15821 3685 1 SYM15890 3694 SYM013333682 0 SYM15890 3694 SYM00741a 3657 1 SYM15890 3694 SYM01315 3677 2SYM15890 3694 SYM15901 3695 3 SYM15890 3694 SYM15870 3692 2 SYM158213685 SYM15870 3692 2 SYM15811 3683 SYM00122 3611 3 SYM15811 3683SYM15890 3694 0 SYM15811 3683 SYM01333 3682 0 SYM15811 3683 SYM013153677 0 SYM15811 3683 SYM00066 3605 0 SYM15811 3683 SYM15870 3692 0SYM01315 3677 SYM15901 3695 0 SYM01315 3677 SYM00124 3613 3

A beneficial plant microbiome is likely made up of multiple strains thatoccupy stress protection niches within the plant. This was evaluated inthe rolling paper assay to test the improvement on the plant phenotypeconferred by inoculation with multiple fungal strains. The top threeperformers all utilize a-Cyclodextrin, which is a trait shared by all ofthe single fungi treatments which incurred the largest positive plantphenotypic change. The majority of the strains that are party of thecombos also show similar patterns when it comes to siderophore and auxinproduction. This may indicate that although they come from differentgenera, they may occupy similar niches when it comes to affecting theplants phenotype when helping the plant deal with drought stress.

Example 10 Trials to Full Plant Maturity to Demonstrate Performance inCommercial Field Setting

Corn was grown at two locations in the United States. Six replicateplots were sown for each treatment and variety combination. Controlplots were planted for formulation treated seeds. Seeds were sown in anirrigated field in plots of 10 by 40 ft arranged in a randomizedcomplete block design. Four rows were planted per plot with a rowspacing of 30 inches. Seeding density at Location 1 was 576 g per acre.Seeding density at Location was 35,000 seeds per acre. SPAD readingswere taken at Location 2 only for 10 plants per plot on three monthsafter planting (one month before harvest) to measure chlorophyll contentof leaves. The interior 2 rows were harvested by combine. Grain yieldper plot, grain moisture, and test weight were assessed. Yield wasadjusted for grain moisture content to a storage moisture of 14% (i.e.dry bushels per acre for combine harvest). Data shown for both locationsare shown in Tables 45A and 45B.

TABLE 45A Rainfed trial in Location 1. Combine yield Treatment (drybu/ac) Bacterial formulation control 101.77 SYM00033 119.96

TABLE 45B Rainfed trial in Location 2. Combine yield Treatment SPAD (drybu/ac) Fungal formulation control 53.72 88.78 SYM00066 ^(i) 54.54 95.17Bacterial formulation control 54.35 91.76 SYM00074 ^(ii) 55.95 85.54^(i) Compare to fungal formulation control ^(ii) Compare to bacterialformulation control

For combine yield, SYM00033 showed a substantial increase in dry bushelsper acre compared to formulation treated controls. As an indicator ofleaf chlorophyll content, SYM00066 showed a slight increase in SPADreadings compared to the fungal formulation control. For combine yield,SYM00066 showed a substantial increase in dry bushels per acre comparedto the fungal formulation control. As an indicator of leaf chlorophyllcontent, SYM00074 showed a slight increase in SPAD readings compared tothe bacterial formulation control.

Other embodiments will be apparent to those skilled in the art fromconsideration of the specification and practice of the embodiments.Consider the specification and examples as exemplary only, with a truescope and spirit being indicated by the following claims.

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REFERENCES

-   Abarenkov, K., R. Henrik Nilsson, K.-H. Larsson, I. J. Alexander, U.    Eberhardt, S. Erland, K. Høiland, R. Kjøller, E. Larsson, T.    Pennanen, R. Sen, A. F. S. Taylor, L. Tedersoo, B. M. Ursing, T.    Vrålstad, K. Liimatainen, U. Peintner, and U. Kõljalg. 2010. The    UNITE database for molecular identification of fungi—recent updates    and future perspectives. New Phytologist 186:281-285.-   Edgar, R. C. 2010. Search and clustering orders of magnitude faster    than BLAST. Bioinformatics 26:2460-2461.-   Edgar, R. C. 2013. UPARSE: highly accurate OTU sequences from    microbial amplicon reads. Nature methods 10:996-8.-   Fierer, N., J. W. Leff, B. J. Adams, U. N. Nielsen, S. T.    Bates, C. L. Lauber, S. Owens, J. a. Gilbert, D. H. Wall, and J. G.    Caporaso. 2012. Cross-biome metagenomic analyses of soil microbial    communities and their functional attributes. Proceedings of the    National Academy of Sciences.-   Lundberg, D. S., S. Yourstone, P. Mieczkowski, C. D. Jones,    and J. L. Dangl. 2013. Practical innovations for high-throughput    amplicon sequencing. Nature methods 10:999-1002.-   McDonald, D., M. N. Price, J. Goodrich, E. P. Nawrocki, T. Z.    DeSantis, A. Probst, G. L. Andersen, R. Knight, and P.    Hugenholtz. 2012. An improved Greengenes taxonomy with explicit    ranks for ecological and evolutionary analyses of bacteria and    archaea. The ISME journal 6:610-8.-   McGuire, K. L., S. G. Payne, M. I. Palmer, C. M. Gillikin, D.    Keefe, S. J. Kim, S. M. Gedallovich, J. Discenza, R. Rangamannar, J.    a Koshner, A. L. Massmann, G. Orazi, A. Essene, J. W. Leff, and N.    Fierer. 2013. Digging the New York City Skyline: soil fungal    communities in green roofs and city parks. PloS one 8:e58020.-   R Core Team. 2013. R: A Language and Environment for Statistical    Computing. R Foundation for Statistical Computing, Vienna, Austria.-   Wang, Q., G. M. Garrity, J. M. Tiedje, and J. R. Cole. 2007. Naive    Bayesian classifier for rapid assignment of rRNA sequences into the    new bacterial taxonomy. Applied and environmental microbiology    73:5261-7.-   Abarenkov, K., R. Henrik Nilsson, K.-H. Larsson, I. J. Alexander, U.    Eberhardt, S. Erland, K. Høiland, R. Kjøller, E. Larsson, T.    Pennanen, R. Sen, A. F. S. Taylor, L. Tedersoo, B. M. Ursing, T.    Vrålstad, K. Liimatainen, U. Peintner, and U. Kõljalg. 2010. The    UNITE database for molecular identification of fungi—recent updates    and future perspectives. New Phytologist 186:281-285.-   Edgar, R. C. 2013. UPARSE: highly accurate OTU sequences from    microbial amplicon reads. Nature methods 10:996-8.-   Fierer, N., J. W. Leff, B. J. Adams, U. N. Nielsen, S. T.    Bates, C. L. Lauber, S. Owens, J. a. Gilbert, D. H. Wall, and J. G.    Caporaso. 2012. Cross-biome metagenomic analyses of soil microbial    communities and their functional attributes. Proceedings of the    National Academy of Sciences.-   Lundberg, D. S., S. Yourstone, P. Mieczkowski, C. D. Jones,    and J. L. Dangl. 2013. Practical innovations for high-throughput    amplicon sequencing. Nature methods 10:999-1002.-   McDonald, D., M. N. Price, J. Goodrich, E. P. Nawrocki, T. Z.    DeSantis, A. Probst, G. L. Andersen, R. Knight, and P.    Hugenholtz. 2012. An improved Greengenes taxonomy with explicit    ranks for ecological and evolutionary analyses of bacteria and    archaea. The ISME journal 6:610-8.-   McGuire, K. L., S. G. Payne, M. I. Palmer, C. M. Gillikin, D.    Keefe, S. J. Kim, S. M. Gedallovich, J. Discenza, R. Rangamannar, J.    a Koshner, A. L. Massmann, G. Orazi, A. Essene, J. W. Leff, and N.    Fierer. 2013. Digging the New York City Skyline: soil fungal    communities in green roofs and city parks. PloS one 8:e58020.-   R Core Team. 2013. R: A Language and Environment for Statistical    Computing. R Foundation for Statistical Computing, Vienna, Austria.-   Rideout J R, He Y, Navas-Molina J A, Walters W A, Ursell L K,    Gibbons S M, Chase J, McDonald D, Gonzalez A, Robbins-Pianka A,    Clemente J C, Gilbert J A, Huse S M, Zhou H, Knight R, Caporaso    J G. (2014) Subsampled open-reference clustering creates consistent,    comprehensive OTU definitions and scales to billions of sequences.    Peed 2:e545https://dx.doi.org/10.7717/peerj.545-   Wang, Q., G. M. Garrity, J. M. Tiedje, and J. R. Cole. 2007. Naive    Bayesian classifier for rapid assignment of rRNA sequences into the    new bacterial taxonomy. Applied and environmental microbiology    73:5261-7.-   Barua, D., Kim, J., & Reed, J. L. (2010) An automated    phenotype-driven approach (GeneForce) for refining metabolic and    regulatory models. PLoS Comput Biol, 6(10), e1000970.-   Blumenstein, K., Albrectsen, B. R., Martin, J. A., Hultberg, M.,    Sieber, T. N., Helander, M., & Witzell, J. (2015) Nutritional niche    overlap potentiates the use of endophytes in biocontrol of a tree    disease. BioControl, 1-13.-   Borglin, S., Joyner, D., DeAngelis, K. M., Khudyakov, J.,    D'haeseleer, P., Joachimiak, M. P., & Hazen, T. (2012) Application    of phenotypic microarrays to environmental microbiology. Current    opinion in biotechnology, 23(1), 41-48.-   Garland, J. L., & Mills, A. L. (1991) Classification and    characterization of heterotrophic microbial communities on the basis    of patterns of community-level sole-carbon-source utilization.    Applied and environmental microbiology, 57(8), 2351-2359.-   Siemens, N., Fiedler, T., Normann, J., Klein, J., Munch, R.,    Patenge, N., & Kreikemeyer, B. (2012) Effects of the ERES    pathogenicity region regulator Ralp3 on Streptococcus pyogenes    serotype M49 virulence factor expression. Journal of bacteriology,    194(14), 3618-3626.-   Chen, T. H., & Murata, N. (2002). Enhancement of tolerance of    abiotic stress by metabolic engineering of betaines and other    compatible solutes. Current opinion in plant biology, 5(3), 250-257.-   Rideout J R, He Y, Navas-Molina J A, Walters W A, Ursell L K,    Gibbons S M, Chase J, McDonald D, Gonzalez A, Robbins-Pianka A,    Clemente J C, Gilbert J A, Huse S M, Zhou H, Knight R, Caporaso    J G. (2014) Subsampled open-reference clustering creates consistent,    comprehensive OTU definitions and scales to billions of sequences.    PeerJ 2:e545 https://dx.doi.org/10.7717/peerj.545

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20160021891A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

We claim:
 1. A method for preparing an agricultural seed composition,comprising contacting a plurality of seeds with a formulation comprisingan effective amount of a synthetic combination of at least twoendophytes heterologously disposed to the plurality of seeds, whereinthe at least two endophytes are selected from genus Cladosporium,Fusarium, Nigrospora, Epicoccum, or Alternaria, and the effective amountincreases tolerance to drought in plants grown from the agriculturalseed compositions, as compared to isoline plants grown from seeds notcontacted with the formulation.
 2. The method of claim 1, wherein atleast one of the endophytes is capable of metabolizing one or more ofD-alanine, D-aspartic acid, D-serine, D-threonine, glycyl-L-asparticacid, glycyl-L-glutamic acid, glycyl-L-proline, glyoxylic acid, inosine,L-alanine, L-alanyl-glycine, L-arabinose, L-asparagine, L-aspartic acid,L-glutamic acid, L-glutamine, L-proline, L-serine, L-threonine,tyramine, uridine, proline, arabinose, xylose, mannose, sucrose,maltose, D-glucosamine, trehalose, oxalic acid, and salicin.
 3. Themethod of claim 1, wherein at least one of the endophytes is capable ofproducing one or more of an auxin, acetoin, or siderophore.
 4. Themethod of claim 1, wherein at least one of the endophytes comprises anucleic acid sequence encoding a protein allowing the endophyte tometabolize arabinose.
 5. The method of claim 4, wherein the proteincomprises a polypeptide sequence selected from the group consisting ofSEQ ID NO: 3701-3813.
 6. A plant derived from the agricultural seedcomposition of claim 1, wherein the plant comprises at least one of theendophytes in at least one of its plant elements.
 7. A progeny of theplant of claim 6, wherein the progeny comprises at least one of theendophytes in at least one of its progeny plant elements.
 8. The methodof claim 1, wherein the seeds are monocot plant seeds.
 9. The method ofclaim 8, wherein the monocot plant is selected from the group consistingof corn, wheat, barley and rice.
 10. The method of claim 1, wherein theseeds are dicot plant seeds.
 11. The method of claim 10, wherein thedicot plant is selected from the group consisting of soybean, canola,cotton, tomato and pepper.
 12. The method of claim 1, wherein the seedsare transgenic seeds.
 13. The method of claim 1, wherein the at leasttwo endophytes are detectable within a target tissue of an agriculturalplant grown from the agricultural seed composition, the target tissueselected from a fruit, seed, leaf, root or portion thereof.
 14. Themethod of claim 1, wherein the at least two endophytes are detected inan amount of at least 100 CFU or spores, or more, in the plurality ofcontacted seeds.
 15. The method of claim 1, wherein the effective amountincreases the biomass, or yield of a fruit or seed produced by a plantgrown from the agricultural seed composition, by at least at least 5%,at least 10%, or more, when compared with the fruit or seed of isolineplants grown from seeds not contacted with the formulation.
 16. Themethod of claim 1, wherein the effective amount increases the rate ofgermination of the agricultural seed composition compared to seeds notcontacted with the formulation.
 17. A method for preparing anagricultural seed composition, comprising contacting a plurality ofseeds with a formulation comprising an effective amount of a syntheticcombination of at least two endophytes heterologously disposed to theplurality of seeds, wherein at least one of the endophytes is capable ofmetabolizing one or more of D-alanine, D-aspartic acid, D-serine,D-threonine, glycyl-L-aspartic acid, glycyl-L-glutamic acid,glycyl-L-proline, glyoxylic acid, inosine, L-alanine, L-alanyl-glycine,L-arabinose, L-asparagine, L-aspartic acid, L-glutamic acid,L-glutamine, L-proline, L-serine, L-threonine, tyramine, uridine,proline, arabinose, xylose, mannose, sucrose, maltose, D-glucosamine,trehalose, oxalic acid, and salicin, and the effective amount increasestolerance to drought in plants grown from the agricultural seedcompositions, as compared to isoline plants grown from seeds notcontacted with said formulation.
 18. A method for preparing anagricultural seed composition, comprising contacting a plurality ofseeds with a formulation comprising an effective amount of a syntheticcombination of at least two endophytes heterologously disposed to theplurality of contacted seeds, wherein at least one endophyte is capableof at least one function or activity selected from the group consistingof: auxin production, nitrogen fixation, production of an antimicrobialcompound, mineral phosphate solubilization, siderophore production,cellulase production, chitinase production, xylanase production, andacetoin production, and wherein the effective amount increases toleranceto drought in plants grown from the agricultural seed compositions, ascompared to isoline plants grown from seeds not contacted with saidformulation.
 19. The method of claim 1, wherein plants grown from theagricultural seed compositions show at least a 5% increase in rootlength when tested in a seedling drought assay, as compared to isolineplants grown from seeds not contacted with said formulation, wherein theseedling drought assay is a seed germination (rolling) paper assaycomprising 8% PEG.
 20. The method of claim 1, wherein plants grown fromthe agricultural seed compositions show at least a 20% growthenhancement when tested in a seedling drought assay, as compared toisoline plants grown from seeds not contacted with said formulation,wherein the seedling drought assay is a filter paper growth assaycomprising 8% PEG.